Neisseria meningitidis compositions and methods thereof

ABSTRACT

In one aspect, the invention relates to a composition including a first polypeptide having the sequence set forth in SEQ ID NO: 1 and a second polypeptide having the sequence set forth in SEQ ID NO: 2. In one embodiment, the composition includes about 120 μg/ml of a first polypeptide including the amino acid sequence set forth in SEQ ID NO: 1, 120 μg/ml of a second polypeptide including the amino acid sequence set forth in SEQ ID NO: 2, about 2.8 molar ratio polysorbate-80 to the first polypeptide, about 2.8 molar ratio polysorbate-80 to the second polypeptide, about 0.5 mg/ml aluminum, about 10 mM histidine, and about 150 mM sodium chloride. In one embodiment, a dose of the composition is about 0.5 ml in total volume. In one embodiment, two-doses of the composition induce a bactericidal titer against diverse heterologous subfamily A and subfamily B strains in a human.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/543,232, filed Jul. 12, 2017, (now allowed), which is a U.S.national phase application under 35 U.S.C. § 371 of InternationalApplication No. PCT/162016/050829, filed Feb. 16, 2016, which claims thebenefit of U.S. Provisional Patent Application 62/118,457, filed on Feb.19, 2015, and U.S. Provisional Patent Application 62/280,212, filed onJan. 19, 2016. All of the foregoing applications are hereby incorporatedby reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to Neisseria meningitidis compositions andmethods thereof.

BACKGROUND OF THE INVENTION

Neisseria meningitidis is a Gram-negative encapsulated bacterium thatcan cause sepsis, meningitis, and death. N. meningitidis can beclassified into at least 12 serogroups (including serogroups A, B, C,29E, H, I, K, L, W-135, X, Y and Z) based on chemically andantigenically distinctive polysaccharide capsules. Strains with five ofthe serogroups (A, B, C, Y, and W135) are responsible for the majorityof disease.

Meningococcal meningitis is a devastating disease that can kill childrenand young adults within hours despite the availability of antibiotics.There is a need for improved immunogenic compositions againstmeningococcal serogroups A, B, C, Y, and W135 and/or X.

Currently, a cross-protective vaccine or composition effective against awide range of MnB isolates is not yet commercially available. Forexample, published results-to-date relating to a licensedmulti-component composition for protection against serogroup B diseasehas not demonstrated a direct bactericidal immune response againstmultiple strains expressing heterologous LP2086 (fHBP) variants, atleast in adolescents. At most, published results-to-date relating to themulti-component composition for protection against serogroup B diseaseappear to show immunogenicity against LP2086 (fHBP) variants that arehomologous to the LP2086 (fHBP) variant in the multi-componentcomposition. Accordingly, a cross-protective vaccine or compositioneffective against diverse MnB isolates is needed as is determiningreal-world vaccine coverage against a panel of diverse or heterologousmeningococcal strains (e.g., representing different geographicalregions).

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a composition including about120 μg/ml of a first lipidated polypeptide including the amino acidsequence set forth in SEQ ID NO: 1, 120 μg/ml of a second lipidatedpolypeptide including the amino acid sequence set forth in SEQ ID NO: 2,about 2.8 molar ratio polysorbate-80 to the first polypeptide, about 2.8molar ratio polysorbate-80 to the second polypeptide, about 0.5 mg/mlaluminum, about 10 mM histidine, and about 150 mM sodium chloride. Inone embodiment, the first dose is about 0.5 ml in total volume. In oneembodiment, the composition induces a bactericidal immune responseagainst N. meningitidis serogroup B. In one embodiment, the compositionmay induce a bactericidal immune response against N. meningitidisserogroup A, C, 29E, H, I, K, L, W-135, X, Y or Z. In one embodiment,the composition does not further include a polypeptide having less than100% sequence identity to SEQ ID NO: 1. In one embodiment, thecomposition does not further include a polypeptide having less than 100%sequence identity to SEQ ID NO: 2. In one embodiment, the firstpolypeptide has a total of 258 amino acids. In one embodiment, thesecond polypeptide has a total of 261 amino acids. In one embodiment,the composition induces a bactericidal titer of serum immunoglobulinagainst N. meningitidis serogroup B that is at least 2-fold higher inthe human after receiving the first dose than a bactericidal titer ofserum immunoglobulin against N. meningitidis serogroup B in the humanprior to receiving the first dose, wherein the increase in bactericidaltiter is measured under identical conditions in a serum bactericidalassay using human complement. In one embodiment, the first lipidatedpolypeptide consists of the amino acid sequence set forth in SEQ IDNO: 1. In one embodiment, the second lipidated polypeptide consists ofthe amino acid sequence set forth in SEQ ID NO: 2.

In another aspect, the invention relates to a method of inducing animmune response against Neisseria meningitidis serogroup B in a human.The method includes administering to the human a first dose and a seconddose of an effective amount of a composition, said composition including120 μg/ml of a first lipidated polypeptide including the amino acidsequence set forth in SEQ ID NO: 1, 120 μg/ml of a second lipidatedpolypeptide including the amino acid sequence set forth in SEQ ID NO: 2,2.8 molar ratio polysorbate-80 to the first polypeptide, 2.8 molar ratiopolysorbate-80 to the second polypeptide, 0.5 mg/ml aluminum, 10 mMhistidine, and 150 mM sodium chloride. In one embodiment, a dose of thecomposition has a total volume of 0.5 ml. In one embodiment, the humanis administered at most two doses of the composition. In one embodiment,the human is not further administered a booster dose of the composition.In one embodiment, the human is administered a third dose of thecomposition. In one embodiment, the human is not further administered abooster dose of the composition after the third dose. In one embodiment,the human is not further administered a fourth dose of the composition.In one embodiment, the third dose is administered to the human within aperiod of about 6 months after the first dose. In one embodiment, thesecond dose is administered at least 30 days after the first dose. Inone embodiment, the method further includes administering a third doseof the composition, wherein the third dose is administered at least 90days after the second dose. In one embodiment, the composition induces abactericidal titer of serum immunoglobulin against N. meningitidisserogroup B that is at least 2-fold higher in the human after receivingthe first dose than a bactericidal titer of serum immunoglobulin againstN. meningitidis serogroup B in the human prior to receiving the firstdose, when measured under identical conditions in a serum bactericidalassay using human complement. In one embodiment, the immune response isbactericidal against a N. meningitidis serogroup B subfamily A strainthat is heterologous to a N. meningitidis strain expressing A05. In oneembodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that is heterologous to a N.meningitidis strain expressing B01. In one embodiment, the immuneresponse is bactericidal against a N. meningitidis serogroup B subfamilyA strain that is heterologous to N. meningitidis strain M98250771. Inone embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that is heterologous to N.meningitidis strain CDC1127. In a preferred embodiment, the immuneresponse is bactericidal against a N. meningitidis serogroup B subfamilyB strain that is heterologous to N. meningitidis strain CDC1573. In oneembodiment, the first polypeptide has a total of 258 amino acids. In oneembodiment, the second polypeptide has a total of 261 amino acids. Inone embodiment, the first lipidated polypeptide consists of the aminoacid sequence set forth in SEQ ID NO: 1. In one embodiment, the secondlipidated polypeptide consists of the amino acid sequence set forth inSEQ ID NO: 2.

In another aspect, the invention relates to a composition that includes60 μg of a first lipidated polypeptide including the amino acid sequenceset forth in SEQ ID NO: 1, 60 μg of a second lipidated polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 2, 2.8 molarratio polysorbate-80 to the first polypeptide, 2.8 molar ratiopolysorbate-80 to the second polypeptide, 0.5 mg/ml aluminum, 10 mMhistidine, and 150 mM sodium chloride, wherein the composition has atotal volume of about 0.5 ml. In one embodiment, the composition inducesa bactericidal immune response against a N. meningitidis serogroup Bsubfamily A strain that is heterologous to a N. meningitidis strainexpressing A05. In one embodiment, the composition induces abactericidal immune response against a N. meningitidis serogroup Bsubfamily B strain that is heterologous to a N. meningitidis strainexpressing B01. In one embodiment, the composition induces abactericidal titer of serum immunoglobulin against N. meningitidisserogroup B that is at least 2-fold higher in the human after receivingthe first dose than a bactericidal titer of serum immunoglobulin againstN. meningitidis serogroup B in the human prior to receiving the firstdose, when measured under identical conditions in a serum bactericidalassay using human complement. In one embodiment, the composition doesnot further include a polypeptide having less than 100% sequenceidentity to SEQ ID NO: 1. In one embodiment, the composition does notfurther include a polypeptide having less than 100% sequence identity toSEQ ID NO: 2. In one embodiment, the first polypeptide has a total of258 amino acids. In one embodiment, the second polypeptide has a totalof 261 amino acids. In one embodiment, the first lipidated polypeptideconsists of the amino acid sequence set forth in SEQ ID NO: 1. In oneembodiment, the second lipidated polypeptide consists of the amino acidsequence set forth in SEQ ID NO: 2.

In a further aspect, the invention relates to a method of inducing abactericidal immune response against a Neisseria meningitidis serogroupB subfamily A strain, and against a Neisseria meningitidis serogroup Bsubfamily B strain in a human. The method includes administering to thehuman an effective amount of a N. meningitidis rLP2086 composition, saidcomposition comprising a) a first lipidated polypeptide comprising theamino acid sequence set forth in SEQ ID NO: 1, and b) a second lipidatedpolypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, wherein the method further comprises administering to the human atleast one of the following immunogenic compositions within 24 hours ofadministering said composition against N. meningitidis serogroup B: (i)an immunogenic composition against a Neisseria meningitidis serogroup Astrain, a Neisseria meningitidis serogroup C strain, a Neisseriameningitidis serogroup Y strain, and/or a Neisseria meningitidisserogroup W135 strain; (ii) an immunogenic composition againstdiphtheria, tetanus, and pertussis; (iii) an immunogenic compositionagainst hepatitis A virus; (iv) an immunogenic composition against humanpapillomavirus; (v) an immunogenic composition against diphtheria,tetanus, pertussis and poliomyelitis; or (vi) any combination thereof.“Immunogenic” refers to an ability to induce an immune response.

In one aspect, the invention relates to a method of inducing abactericidal immune response against a N. meningitidis serogroup Bsubfamily A strain, and against a N. meningitidis serogroup B subfamilyB strain in a human. The method includes administering to the human aneffective amount of a N. meningitidis rLP2086 composition, saidcomposition including a) a first lipidated polypeptide including theamino acid sequence set forth in SEQ ID NO: 1, and b) a second lipidatedpolypeptide including the amino acid sequence set forth in SEQ ID NO: 2,wherein the method further includes concomitantly administering to thehuman at least one of the following additional immunogenic compositions:(i) an immunogenic composition against a N. meningitidis serogroup Astrain, a N. meningitidis serogroup C strain, a N. meningitidisserogroup Y strain, and/or a N. meningitidis serogroup W135 strain; (ii)an immunogenic composition against diphtheria, tetanus, and pertussis;(iii) an immunogenic composition against hepatitis A virus; (iv) animmunogenic composition against human papillomavirus; (v) an immunogeniccomposition against diphtheria, tetanus, pertussis and poliomyelitis; or(vi) any combination thereof.

In another aspect, the invention relates to use of an effective amountof a N. meningitidis rLP2086 composition and at least one additionalimmunogenic composition concomitantly for inducing a bactericidal immuneresponse against a Neisseria meningitidis serogroup B subfamily Astrain, and against a Neisseria meningitidis serogroup B subfamily Bstrain in a human, wherein said N. meningitidis rLP2086 compositioncomprises a) a first lipidated polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 1, and b) a second lipidatedpolypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, wherein the at least one additional immunogenic composition is: (i)an immunogenic composition against a Neisseria meningitidis serogroup Astrain, a Neisseria meningitidis serogroup C strain, a Neisseriameningitidis serogroup Y strain, and/or a Neisseria meningitidisserogroup W135 strain; (ii) an immunogenic composition againstdiphtheria, tetanus, and pertussis; (iii) an immunogenic compositionagainst hepatitis A virus; (iv) an immunogenic composition against humanpapillomavirus; (v) an immunogenic composition against diphtheria,tetanus, pertussis and poliomyelitis; or (vi) any combination thereof.

In one embodiment, the N. meningitidis rLP2086 composition furtherincludes polysorbate-80. In one embodiment, the N. meningitidis rLP2086composition further includes aluminum. In one embodiment, the N.meningitidis rLP2086 composition further includes histidine buffer. Inone embodiment, the N. meningitidis rLP2086 composition further includessodium chloride. In one embodiment, the N. meningitidis rLP2086composition includes about 120 μg/ml of the first polypeptide; about 120μg/ml of the second polypeptide; about 2.8 molar ratio ofpolysorbate-80; about 0.5 mg/ml aluminum; about 10 mM histidine; andabout 150 mM sodium chloride. In one embodiment, the N. meningitidisrLP2086 composition includes about 60 μg of the first polypeptide; about60 μg of the second polypeptide; about 18 μg polysorbate-80; about 250μg aluminum; about 780 μg histidine; and about 4380 μg sodium chloride.

In one embodiment, the N. meningitidis rLP2086 composition induces abactericidal immune response against at least one of N. meningitidisserogroup B A22, A56, B24, B44 strains, or any combination thereof. Inone embodiment, the N. meningitidis rLP2086 composition induces abactericidal immune response against N. meningitidis serogroup B A22. Inone embodiment, the N. meningitidis rLP2086 composition induces abactericidal immune response against N. meningitidis serogroup B B24. Inone embodiment, the N. meningitidis rLP2086 composition induces abactericidal immune response against at least one of N. meningitidisserogroup B B24, B16, B44, A22, B03, B09, A12, A19, A05, A07, B153strains, or any combination thereof.

In one embodiment, the method further includes inducing an immuneresponse against at least one of a Neisseria meningitidis serogroup Astrain, a Neisseria meningitidis serogroup C strain, a Neisseriameningitidis serogroup Y strain, and/or a Neisseria meningitidisserogroup W135 strain, or any combination thereof.

In one embodiment, the method includes administering to the human anadditional immunogenic composition including a mixture of four distinctand separately made protein-capsular polysaccharide conjugates, whereinthe first conjugate includes purified N. meningitidis capsularpolysaccharide of serogroup W-135 conjugated to a carrier protein, thesecond conjugate includes purified N. meningitidis capsularpolysaccharide of serogroup Y conjugated to a carrier protein, the thirdconjugate includes purified N. meningitidis capsular polysaccharide ofserogroup A conjugated to a purified carrier protein, and the fourthconjugate includes purified N. meningitidis capsular polysaccharide ofserogroup C conjugated to a carrier protein, wherein the carrier proteinis selected from the group consisting of diphtheria toxoid, CRM197, andtetanus toxoid. In one embodiment, the carrier protein is diphtheriatoxoid.

In one embodiment, the immunogenic composition is a liquid composition.

In one embodiment, the immunogenic composition is not lyophilized.

In one embodiment, the method further includes inducing an immuneresponse against at least one of human papillomavirus type 6, 11, 16,18, or any combination thereof. In one embodiment, the method furtherincludes inducing an immune response against at least one of diphtheria,tetanus, pertussis, poliomyelitis, or any combination thereof in thehuman.

In one embodiment, the effective amount of the N. meningitidis rLP2086composition includes one dose. In one embodiment, the effective amountof the N. meningitidis rLP2086 composition includes two doses. In oneembodiment, the effective amount of the N. meningitidis rLP2086composition further includes a booster dose. In one embodiment, theeffective amount of the N. meningitidis rLP2086 composition includes atmost three doses.

In one embodiment, the N. meningitidis rLP2086 composition isadministered within 24 hours of administration of the additionalcomposition. In one embodiment, the N. meningitidis rLP2086 compositionis simultaneously administered to the additional composition. In oneembodiment, a first dose of the N. meningitidis rLP2086 composition isconcomitantly administered to the additional composition. In oneembodiment, the first dose of the N. meningitidis rLP2086 composition issimultaneously administered to the additional composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Proportion of Subjects Achieving hSBA Titers ωLLOQ. hSBA=serumbactericidal assay using human complement; LLOQ=lower limit ofquantitation.

FIG. 2—Percentage of subjects achieving 4× rise in hSBA titers toPrinceton University Outbreak Strains and UCSB Outbreak Strains ofIndividual Human Subjects Following Immunization With rLP2086 (StudyB1971012—described in Example 5, Example 6). Serum samples from ninehuman subjects immunized with bivalent rLP2086 in clinical studyB1971012 were evaluated in exploratory hSBAs using MnB outbreak strainsfrom Princeton University and from UCSB. See Example 9.

SEQUENCE IDENTIFIERS

SEQ ID NO: 1 sets forth the amino acid sequence for a recombinant N.meningitidis, serogroup B, 2086 variant A05 polypeptide antigen.

SEQ ID NO: 2 sets forth the amino acid sequence for a recombinant N.meningitidis, serogroup B, 2086 variant B01 polypeptide antigen.

SEQ ID NO: 3 sets forth the amino acid residues at positions 1-4 of SEQID NO: 1 and SEQ ID NO: 2.

SEQ ID NO: 4 sets forth the amino acid sequence of the N-terminus of arecombinant Neisserial Subfamily A LP2086 polypeptide (rLP2086) (A05)polypeptide antigen.

SEQ ID NO: 5 sets forth the amino acid sequence of the N-terminus ofNeisserial Subfamily A LP2086 M98250771 polypeptide (A05) polypeptideantigen.

SEQ ID NO: 6 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B153.

SEQ ID NO: 7 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A04.

SEQ ID NO: 8 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A05

SEQ ID NO: 9 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A12.

SEQ ID NO: 10 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A22.

SEQ ID NO: 11 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B02.

SEQ ID NO: 12 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B03.

SEQ ID NO: 13 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B09.

SEQ ID NO: 14 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B22.

SEQ ID NO: 15 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B24.

SEQ ID NO: 16 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B44.

SEQ ID NO: 17 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B16.

SEQ ID NO: 18 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A07.

SEQ ID NO: 19 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A19.

SEQ ID NO: 20 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A06.

SEQ ID NO: 21 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A15.

SEQ ID NO: 22 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A29.

SEQ ID NO: 23 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B15.

DETAILED DESCRIPTION OF THE INVENTION

The inventors surprisingly discovered an rLP2086 composition thatincludes a first lipidated polypeptide including the amino acid sequenceset forth in SEQ ID NO: 1 and a second lipidated polypeptide includingthe amino acid sequence set forth in SEQ ID NO: 2. The composition hasan acceptable safety profile in humans and the composition surprisinglyelicits a broadly cross-reactive bactericidal immune response in humansagainst at least more than two diverse Neisseria meningitidis strains.

The inventors also discovered that robust immune responses against N.meningitidis serogroup B were generated even after concomitantadministration of the rLP2086 composition and at least one of thefollowing immunogenic compositions: (i) an immunogenic compositionagainst a N. meningitidis serogroup A strain, a N. meningitidisserogroup C strain, a N. meningitidis serogroup Y strain, and/or a N.meningitidis serogroup W135 strain; (ii) an immunogenic compositionagainst diphtheria, tetanus, and pertussis; (iii) an immunogeniccomposition against hepatitis A virus; (iv) an immunogenic compositionagainst human papillomavirus; (v) an immunogenic composition againstdiphtheria, tetanus, pertussis and poliomyelitis; or (vi) anycombination thereof.

The concomitant administration of at least the rLP2086 composition and ameningococcal Groups A, C, Y and W-135 polysaccharide conjugate vaccine(MCV4) also surprisingly generated an immune response at least againstmeningococcal Groups A, C, Y and W-135 in addition to meningococcalGroup B. The immune responses against the meningococcal Groups A, C, Yand W-135 after concomitant administration of the rLP2086 compositionand the MCV4 composition were noninferior when compared to the immuneresponse generated by an administration of the MCV4 composition in theabsence of the rLP2086 composition.

The concomitant administration of at least the rLP2086 composition, ameningococcal Groups A, C, Y and W-135 polysaccharide conjugate vaccine(MCV4), and a tetanus, diphtheria, and acellular pertussis (Tdap)vaccine also surprisingly generated an immune response at least againsttetanus, diphtheria, and acellular pertussis in addition tomeningococcal Groups A, C, Y and W-135 and to meningococcal Group B. Theimmune responses against the Tdap and meningococcal Groups A, C, Y andW-135 after concomitant administration of the rLP2086 composition, theMCV4 composition, and Tdap composition were noninferior when compared tothe immune response generated by an administration of the MCV4composition and Tdap composition in the absence of the rLP2086composition.

Moreover, the inventors discovered that the rLP2086 composition can besafely administered to humans concomitantly with an immunogeniccomposition against hepatitis A virus.

The inventors further discovered that a 2-dose administration scheduleand a 3-dose administration schedule surprisingly yielded hSBA titers ofA against test strains from N. meningitidis serogroup B, with vaccineheterologous LP2086 (factor H binding protein (fHBP)) subfamilies A andB in a high proportion of human subjects. A 3-dose administrationschedule may provide broader protection in humans against diverse MnBclinical strains, when compared to a 2-dose administration schedule.

The inventors also surprisingly discovered that robust immune responsesagainst human papillomavirus and N. meningitidis serogroup B weregenerated after concomitant administration of the rLP2086 compositionand a quadrivalent

immunogenic composition against human papillomavirus (HPV4). Forexample, a concomitant administration of the rLP2086 composition andHPV4 composition generated an immune response at least against N.meningitidis serogroup B test strains expressing fHBPs that areheterologous to those fHBPs in the rLP2086 composition. Suchheterologous test strains include wild-type N. meningitidis serogroup Bstrains that express A22 fHBP, A56 fHBP, B24 fHBP, or B44 fHBP, whichare each heterologous to the fHBPs in the rLP2086 composition. SeeWO/2012/032489, WO/2013/132452, US patent publication numberUS20120093852, and US patent publication number US20130243807, whichdescribe variant fHBP proteins, including A22 fHBP, A56 fHBP, B24 fHBP,and B44 fHBP, among others. These references are each incorporated byreference in their entirety. The concomitant administration alsosurprisingly generated an immune response at least against HPV types 6,11, 16, and/or 18. The immune responses against the HPV types afterconcomitant administration of the rLP2086 composition and the HPV4composition were noninferior when compared to the immune responsegenerated by an administration of the HPV4 composition in the absence ofthe rLP2086 composition.

In addition, the inventors surprisingly discovered that robust immuneresponses against diphtheria, tetanus, pertussis and poliomyelitis andN. meningitidis serogroup B were generated after concomitantadministration of the rLP2086 composition and an immunogenic compositionagainst diphtheria, tetanus, pertussis and poliomyelitis. For example, aconcomitant administration of the rLP2086 composition and REPEVAXcomposition generated an immune response at least against N.meningitidis serogroup B test strains expressing fHBPs that areheterologous to those fHBPs in the rLP2086 composition. The concomitantadministration also surprisingly generated an immune response at leastagainst the 9 antigens in REPEVAX: diphtheria, tetanus, pertussistoxoid, pertussis filamentous hemagglutinin, pertussis pertactin,pertussis fimbrial agglutinogens type 2+3, poliovirus type 1, poliovirustype 2, poliovirus type 3. The immune responses against the REPEVAXantigens after concomitant administration of the rLP2086 composition andthe REPEVAX composition were noninferior when compared to the immuneresponse generated by an administration of the REPEVAX composition inthe absence of the rLP2086 composition.

Composition and Vaccine

In one aspect, the invention relates to a composition against Neisseriameningitidis. The composition includes a first lipidated polypeptidehaving the amino acid sequence set forth in SEQ ID NO: 1, and a secondlipidated polypeptide having the amino acid sequence set forth in SEQ IDNO: 2.

The inventors surprisingly discovered a single N. meningitidispolypeptide component that induces an effective broadly protectiveimmune response against multiple strains of N. meningitidis serogroup B.Accordingly, in one embodiment, the composition does not include afusion protein. In one embodiment, the composition does not include achimeric protein. In one embodiment, the composition does not include ahybrid protein. In one embodiment, the composition does not furtherinclude a peptide fragment. In another embodiment, the composition doesnot further include a Neisserial polypeptide that is not fHBP. Forexample, in one embodiment, the composition does not include a PorAprotein. In another embodiment, the composition does not include a NadAprotein. In another embodiment, the composition does not further includea Neisserial heparin binding antigen (NHBA). In another embodiment, thecomposition does not further include a Neisserial outer membrane vesicle(OMV). In a preferred embodiment, the composition does not furtherinclude antigens, other than the first polypeptide and the secondpolypeptide.

In another aspect, the inventors surprisingly discovered thatpolypeptide antigens derived from at most two N. meningitidis serogroupB strains induces an immune response against multiple strains of N.meningitidis serogroup B. Accordingly, in one embodiment, thecomposition does not further include a polypeptide that is not derivedfrom N. meningitidis serogroup B subfamily A M98250771 strain and/or N.meningitidis serogroup B subfamily B CDC1573 strain.

In one embodiment, the composition does not further include apolypeptide having less than 100% sequence identity to SEQ ID NO: 1. Inanother embodiment, the composition does not further include apolypeptide having less than 100% sequence identity to SEQ ID NO: 2. Forexample, the composition does not further include a polypeptide havingless than 100% sequence identity to the full length of SEQ ID NO: 1and/or SEQ ID NO: 2.

In one embodiment, the composition further includes polysorbate-80,aluminum, histidine, and sodium chloride. In one embodiment, thecomposition includes about 60 μg of a first lipidated polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 1, about 60 μgof a second lipidated polypeptide including the amino acid sequence setforth in SEQ ID NO: 2, 2.8 molar ratio of polysorbate-80 to eachpolypeptide, 0.5 mg aluminum/ml as aluminum phosphate, 10 mM histidine,and 150 mM sodium chloride, wherein the composition preferably has atotal volume of about 0.5 ml.

In another aspect, the composition includes about 120 μg/ml of a firstlipidated polypeptide including the amino acid sequence set forth in SEQID NO: 1, about 120 μg/ml of a second lipidated polypeptide includingthe amino acid sequence set forth in SEQ ID NO: 2, 2.8 molar ratio ofpolysorbate-80 to each polypeptide, 0.5 mg aluminum/ml as aluminumphosphate, 10 mM histidine, and 150 mM sodium chloride.

In a further aspect, the composition includes a) 60 μg of a firstlipidated polypeptide including the amino acid sequence set forth in SEQID NO: 1; b) 60 μg of a second lipidated polypeptide including the aminoacid sequence set forth in SEQ ID NO: 2; c) 18 μg polysorbate-80 d) 250μg aluminum; e) 780 μg histidine, and; f) 4380 μg sodium chloride.

In an exemplary embodiment, the composition includes about 60 μg of afirst lipidated polypeptide consisting of the amino acid sequence setforth in SEQ ID NO: 1, about 60 μg of a second lipidated polypeptideconsisting of the amino acid sequence set forth in SEQ ID NO: 2, 2.8molar ratio of polysorbate-80 to first lipidated polypeptide and tosecond lipidated polypeptide, 0.5 mg/ml aluminum phosphate, 10 mMhistidine, and 150 mM sodium chloride, wherein the composition has atotal volume of about 0.5 ml. In the exemplary embodiment, thecomposition is a sterile isotonic buffered liquid suspension. In theexemplary embodiment, the composition has a pH 6.0. In the exemplaryembodiment, the first polypeptide and the second polypeptide areadsorbed to aluminum.

In one embodiment, the composition has a total volume of about 0.5 ml.In one embodiment, a first dose of the composition has a total volume ofabout 0.5 ml. A “first dose” refers to the dose of the composition thatis administered on Day 0. A “second dose” or “third dose” refers to thedose of the composition that is administered subsequently to the firstdose, which may or may not be the same amount as the first dose.

The composition is immunogenic after administration of a first dose to ahuman. In one embodiment, the first dose is about 0.5 ml in totalvolume.

The composition induces a bactericidal titer of serum immunoglobulinagainst N. meningitidis serogroup B that is at least greater than 1-foldhigher, preferably at least 2-fold higher, in the human after receivingthe first dose than a bactericidal titer of serum immunoglobulin againstN. meningitidis serogroup B in the human prior to receiving the firstdose, when measured under identical conditions in a serum bactericidalassay using human complement (hSBA).

In a preferred embodiment, the bactericidal titer or bactericidal immuneresponse is against a N. meningitidis serogroup B subfamily A strain andagainst a N. meningitidis serogroup B subfamily B strain. Mostpreferably, the bactericidal titer or bactericidal immune response is atleast against N. meningitidis serogroup B, subfamily B, B01 strain.

In another preferred embodiment, the bactericidal titer or bactericidalimmune response is at least against N. meningitidis serogroup B,subfamily B, B24 strain. In another preferred embodiment, thebactericidal titer or bactericidal immune response is at least againstN. meningitidis serogroup B, subfamily A, A22 strain.

In one embodiment, the composition induces a bactericidal titer of serumimmunoglobulin against N. meningitidis serogroup B that is at leastgreater than 1-fold, such as, for example, at least 1.01-fold, 1.1-fold,1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or 16-foldhigher in the human after receiving a dose of the composition than abactericidal titer of serum immunoglobulin against N. meningitidisserogroup B in the human prior to receiving said dose, when measuredunder identical conditions in a serum bactericidal assay using humancomplement.

In one embodiment, the composition is an immunogenic composition for ahuman. In another embodiment, the composition is a vaccine. A “vaccine”refers to a composition that includes an antigen, which contains atleast one epitope that induces an immune response that is specific forthat antigen. The vaccine may be administered directly into the subjectby subcutaneous, oral, oronasal, or intranasal routes of administration.Preferably, the vaccine is administered intramuscularly. In oneembodiment, the composition is a vaccine for humans. In one embodiment,the composition is an immunogenic composition against N. meningitidis.

In one embodiment, the composition is a liquid composition. In apreferred embodiment, the composition is a liquid suspensioncomposition. In another preferred embodiment, the composition is notlyophilized.

First Polypeptide

In one embodiment, the composition includes a first polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 1. In one preferredembodiment, the composition includes about 60 μg of a first polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 1, wherein thecomposition preferably has a total volume of 0.5 ml. In anotherembodiment, the composition includes about 120 μg/ml of a firstpolypeptide including the amino acid sequence set forth in SEQ ID NO: 1.The polypeptide is a modified factor H binding protein (fHBP) from N.meningitidis strain M98250771. A description of fHBP is disclosed inWO2012032489 and US patent publication US 2012/0093852, which are eachincorporated by reference in their entirety. The polypeptide isN-terminally lipidated with three predominant fatty acids C16:0, C16:1,and C18:1 covalently linked at three positions of the polypeptide. Thefirst polypeptide includes a total of 258 amino acids.

The first polypeptide includes two modifications introduced in theN-terminal region of the polypeptide, as compared to the correspondingwild-type sequence from N. meningitidis strain M98250771. A glycine inthe second position is added as a consequence of introducing a cloningsite. A second modification includes the deletion of four amino acids.Accordingly, in one embodiment, the first polypeptide includes a C-G-S-Ssequence (SEQ ID NO: 3) at the N-terminus. See SEQ ID NO: 1, first fouramino acid residues.

The N-terminal differences between the first polypeptide sequence andthe wild-type Neisserial sequence is shown below. Accordingly, in oneembodiment, the first polypeptide includes at least the first 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or more amino acid residues of the aminoacid sequence set forth in SEQ ID NO: 1. Preferably, the firstpolypeptide includes at least the first 4, more preferably at least thefirst 6, and most preferably, at least the first 8 amino acid residuesof SEQ ID NO: 1.

Comparison of Predicted N-Terminal Sequences of Recombinant and Neisserial Subfamily A  LP2086 PolypeptiderLP2086 M98250771 CGSS-----GGGGVAAD (SEQ ID NO: 4) Neisserial LP2086 C-SSGS-GSGGGGVAAD (SEQ ID NO: 5) M98250771 >A05  (SEQ ID NO: 1)CGSSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIREKVH EIGIAGKQ

In one embodiment, the first polypeptide includes the amino acidsequence set forth in SEQ ID NO: 1. In one embodiment, the firstpolypeptide has a total of 258 amino acids. In one embodiment, the firstpolypeptide does not include an amino acid sequence having less than100% sequence identity to SEQ ID NO: 1. In another embodiment, the firstpolypeptide consists of the amino acid sequence set forth in SEQ IDNO: 1. In another embodiment, the first polypeptide includes the aminoacid sequence KDN. See for example, amino acid residues 73-75 of SEQ IDNO: 1. In another embodiment, the first polypeptide includes the aminoacid sequence set forth in SEQ ID NO: 3 at the N-terminus of thepolypeptide. In another embodiment, the first polypeptide includes theamino acid sequence set forth in SEQ ID NO: 4 at the N-terminus of thepolypeptide.

In a preferred embodiment, the first polypeptide is readily expressed ina recombinant host cell using standard techniques known in the art. Inanother preferred embodiment, the first polypeptide includes abactericidal epitope on the N- and/or C-domain of SEQ ID NO: 1. In oneembodiment, the first polypeptide includes at least the first 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100 amino acid residues of the amino acid sequence set forthin SEQ ID NO: 1. Preferably, the first polypeptide includes at least thefirst 2, more preferably at least the first 4, and most preferably, atleast the first 8 amino acid residues of SEQ ID NO: 1.

In another embodiment, the first polypeptide includes at least the last4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100 amino acid residues of the amino acidsequence set forth in SEQ ID NO: 1.

Second Polypeptide

In one embodiment, the composition includes a second polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 2. In one preferredembodiment, the composition includes about 60 μg of a second polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 2, wherein thecomposition preferably has a total volume of 0.5 ml. In anotherembodiment, the composition includes 120 μg/ml of a second polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 2. Thepolypeptide is a factor H binding protein (fHBP) from N. meningitidisstrain CDC1573. A description of fHBP is disclosed in WO2012032489 andUS patent publication US 2012/0093852, which are each incorporated byreference in their entirety. The polypeptide is N-terminally lipidatedwith three predominant fatty acids C16:0, C16:1, and C18:1 covalentlylinked at three positions of the polypeptide. The second polypeptideincludes a total of 261 amino acids. In one embodiment, the secondpolypeptide includes a C-G-S-S sequence (SEQ ID NO: 3) at theN-terminus. See the first four amino acid residues of SEQ ID NO: 2.

>B01  (SEQ ID NO: 2) CGSSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVAKRRFRIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAVAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGEKAQEVAGSAEVETAN GIHHIGLAAKQ

In one embodiment, the second polypeptide includes the amino acidsequence set forth in SEQ ID NO: 2. In one embodiment, the secondpolypeptide has a total of 261 amino acids. In one embodiment, thesecond polypeptide consists of the amino acid sequence set forth in SEQID NO: 2. In another embodiment, the second polypeptide does not furtherinclude a polypeptide having less than 100% sequence identity to SEQ IDNO: 2. In a preferred embodiment, the first polypeptide and the secondpolypeptide includes a C-G-S-S(SEQ ID NO: 3) sequence at the N-terminusof the respective polypeptide.

In a preferred embodiment, the second polypeptide is readily expressedin a recombinant host cell using standard techniques known in the art.In another preferred embodiment, the second polypeptide includes abactericidal epitope on the N- and/or C-domain of SEQ ID NO: 2. In oneembodiment, the second polypeptide includes at least the first 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100 amino acid residues of the amino acid sequenceset forth in SEQ ID NO: 2. Preferably, the second polypeptide includesat least the first 2, more preferably at least the first 4, and mostpreferably, at least the first 8 amino acid residues of SEQ ID NO: 2.

In another embodiment, the first polypeptide includes at least the last4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100 amino acid residues of the amino acidsequence set forth in SEQ ID NO: 2.

Polysorbate-80

Polysorbate 80 (PS-80) is a non-ionic surfactant. Accelerated stabilitystudies using an in vitro monoclonal antibody based potency assaydemonstrated instability of the subfamily B protein at higher molarratios of PS-80 to MnB rLP2086 protein in the final formulation. Furtherexperiments with varying ratios of PS-80 have demonstrated that theoptimal molar ratio of PS-80 to MnB rLP2086 protein is approximately2.8±1.4 to retain potency.

The concentration of PS-80 in the composition is dependent on a molarratio of PS-80 to the polypeptide. In one embodiment, the compositionincludes a 2.8±1.4 molar ratio of PS-80 to the first polypeptide and tothe second polypeptide. In one embodiment, the composition includes a2.8±1.1 molar ratio of PS-80 to the first polypeptide and to the secondpolypeptide. In one embodiment, the composition includes at least 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, or 3.3molar ratio of PS-80 to polypeptide. Preferably, the compositionincludes a 2.8 molar ratio of PS-80 to polypeptide.

The PS-80 to polypeptide molar ratio is determined by calculation fromthe measured concentration of PS-80 and the measured total polypeptideconcentration, in which both values are expressed in moles. For example,PS-80 to Protein molar ratio is determined by calculation of themeasured concentration of PS-80 (e.g., by reverse phase high pressureliquid chromatography (RP-HPLC)) to the measured total proteinconcentration (e.g., by ion exchange-high pressure liquid chromatography(IEX-HPLC)) in the final drug substance, where both values are expressedin moles.

A RP-HPLC is used to quantitate the concentration of Polysorbate 80 invaccine formulations. The concentration of detergent is determined bysaponification of the fatty acid moiety; Polysorbate 80 is converted tofree oleic acid by alkaline hydrolysis at 40° C. The sample is separatedby RP-HPLC using a C18 column and quantitated using a UV detector at awavelength of 200 nm.

The first and the second polypeptides are resolved by anion-exchangeHPLC. rLP2086(fHBP) Subfamily A and B proteins elute at distinctretention times and are quantitated using a standard curve generatedagainst the respective rLP2086 protein reference material.

The term “molar ratio” and a description of an immunogenic compositionincluding a fHBP and PS-80 is further disclosed in WO2012025873 and USpatent publication US 2013/0171194, which are each incorporated byreference in their entirety.

The term “molar ratio” as used herein refers to the ratio of the numberof moles of two different elements in a composition. In someembodiments, the molar ratio is the ratio of moles of detergent to molesof polypeptide. In some embodiments, the molar ratio is the ratio ofmoles of PS-80 to moles of protein. In one embodiment, based on theprotein and Polysorbate 80 concentrations, the Molar Ratio may becalculated using the following equation:

${{Molar}\mspace{14mu} {Ratio}} = {\frac{\% \mspace{14mu} {PS}\text{-}80}{{mg}\text{/}{ml}\mspace{14mu} {protein}} \times 216}$

In one embodiment, the composition includes about 0.0015, 0.0017,0.0019, 0.0021, 0.0023, 0.0025, 0.0027, 0.0029, 0.0031, 0.0033, 0.0035,0.0037, 0.0039, 0.0041, 0.0043, 0.0045, 0.0047, 0.0049, 0.0051 mg/mLPS-80. Preferably, the composition includes about 0.0035 mg/mL PS-80.

In another embodiment, the composition includes about 10 μg, 11 μg, 12μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22μg, 23 μg, 24 μg, or 25 μg PS-80. In a preferred embodiment, thecomposition includes about 18 μg PS-80.

In another embodiment, the composition includes a PS-80 concentrationranging from 0.0005% to 1%. For example, the PS-80 concentration in thecomposition may be at least 0.0005%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%,0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,0.7%, 0.8%, 0.9%, 1%, or 1.1% PS-80.

In a preferred embodiment, the composition includes about 0.07% PS-80.

Any Minimum Value May be Combined with any Maximum Value DescribedHerein to Define a Range. Aluminum

The composition preferably includes about 0.5 mg/ml aluminum phosphate.In one embodiment, the composition includes about 0.5 mg aluminum/ml asaluminum phosphate. AlPO₄ at 0.50 mg/ml is added as a stabilizer toprovide enhanced manufacturability and stability. This concentrationmaintains binding (90% binding or better) of the subfamily A and Bproteins to aluminum.

The process for producing an aluminum phosphate is described in USpatent publication US 2009/0016946, which is incorporated by referencein its entirety.

In one embodiment, the composition does not further include amultivalent cation, other than aluminum. In one embodiment, thecomposition does not further include Al(OH)₃ or Al(SO₄)₃.

Excipients

In one embodiment, the composition includes histidine. In oneembodiment, the composition includes sodium chloride. The compositionpreferably includes about 10 mM histidine, and about 150 mM sodiumchloride. In one embodiment, the composition includes 10 mM histidineand 150 mM sodium chloride.

In another embodiment, the composition includes about 650 μg, 660 μg,670 μg, 680 μg, 690 μg, 700 μg, 710 μg, 720 μg, 730 μg, 740 μg, 750 μg,760 μg, 770 μg, 780 μg, 790 μg, 800 μg, 810 μg, 820 μg, 830 μg, 840 μg,or 850 μg of histidine. Preferably, the composition includes about 780μg histidine. Any minimum value may be combined with any maximum valuedescribed herein to define a range.

In one embodiment, the composition includes a tris, phosphate, orsuccinate buffer. In a preferred embodiment, the composition does notinclude tris buffer. In a preferred, the composition does not includephosphate buffer. In one preferred embodiment, the composition does notinclude succinate buffer. In a preferred embodiment, the compositionincludes histidine buffer.

In a preferred embodiment, the pH of the composition is between 6.0 and7.0, most preferably pH 6.0. In one embodiment, the pH of thecomposition is at most 6.1.

Bactericidal Activity

Immune response induced by administering the composition to a human isdetermined using a serum bactericidal assay using human complement(hSBA) against four N. meningitidis serogroup B (MnB) strains. The 4 MnBstrains used in the hSBA were selected from a strain pool. The strainpool represented a collection of systematically collected clinicallyrelevant N. meningitidis serogroup B strains from the US and Europe. Twoof the 4 strains for the SBA are from N. meningitidis serogroup B LP2086(fHBP) subfamily A, and another two of the 4 strains are from N.meningitidis serogroup B LP2086(fHBP) subfamily B.

The high proportion of hSBA response to all test strains, especiallystrains expressing lipoprotein 2086 variants with sequences heterologousto the first polypeptide suggests that the composition is a broadlyprotective vaccine and that two doses are sufficient to confer highseroprotection at least against N. meningitidis serogroup B subfamily Astrains.

The high proportion of hSBA response to all test strains, especiallystrains expressing lipoprotein 2086 variants with sequences heterologousto both the first polypeptide and the second polypeptide suggests thatthe composition is a broadly protective vaccine and that at most threedoses within about a 6 month period are sufficient to confer highseroprotection against N. meningitidis serogroup B strains expressingrLP2086 (FHBP) subfamily A and/or subfamily B.

In one embodiment, the hSBA strain is an LP2086 (fHBP) subfamily Astrain. In one embodiment, the hSBA strain is an LP2086 (fHBP) subfamilyA strain that expresses a lipoprotein 2086 variant that is heterologousto a N. meningitidis strain expressing A05. For example, in oneembodiment, the hSBA strain is an LP2086 (fHBP) subfamily A strain thatexpresses a lipoprotein 2086 variant that is heterologous to strainM98250771. In one embodiment, the hSBA strain is an LP2086 (fHBP) A22strain. In another embodiment, the hSBA strain is an LP2086 (fHBP) A56strain. In a further embodiment, the hSBA strains are LP2086 (fHBP) A22and LP2086 (fHBP) A56 strains. In another embodiment, the hSBA strain isan LP2086 A04 strain. In one embodiment, the hSBA strain is an LP2086A05 strain. In one embodiment, the hSBA strain is an LP2086 A12 strain.In one embodiment, the hSBA strain is an LP2086 A22 strain. In oneembodiment, the hSBA strain is an LP2086 A12 strain. In one embodiment,the hSBA strain is an LP2086 A04 strain. In one embodiment, the hSBAstrain is an LP2086 Al 9 strain. In one embodiment, the hSBA strain isan LP2086 A07 strain. In a further embodiment, the hSBA strains includeA22, A12, A19, A05, and A07, or any combination thereof. In oneembodiment, the hSBA strains include A06, A15, and A29, or anycombination thereof.

In one embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that is heterologous to a N.meningitidis strain expressing A05. In one embodiment, the immuneresponse is against N. meningitidis serogroup B A22 strain. In oneembodiment, the immune response is against N. meningitidis serogroup BA56 strain. In one embodiment, the immune response is against N.meningitidis serogroup B A06 strain. In one embodiment, the immuneresponse is against N. meningitidis serogroup B A15 strain. In oneembodiment, the immune response is against N. meningitidis serogroup BA29 strain. In one embodiment, the immune response is against N.meningitidis serogroup B A62 strain. In one embodiment, the immuneresponse is bactericidal against a N. meningitidis serogroup B subfamilyA strain that is heterologous to N. meningitidis strain M98250771. Inone embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity to the first polypeptide. Inanother embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity to a factor H binding proteinexpressed by N. meningitidis strain M98250771. In a preferredembodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at least 80%,more preferably at least 84%, identity to a factor H binding proteinexpressed by N. meningitidis strain M98250771.

In another embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at most 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identity to the first polypeptide. Inanother embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at most 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identity to a factor H binding proteinexpressed by N. meningitidis strain M98250771. In a preferredembodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at most 99%,more preferably at most 85%, identity to a factor H binding proteinexpressed by N. meningitidis strain M98250771. Any minimum value may becombined with any maximum value described herein to define a range.

In one embodiment, the hSBA strain is an LP2086 (fHBP) subfamily Bstrain. In one embodiment, the hSBA strain is an LP2086 (fHBP) subfamilyB strain that expresses a lipoprotein 2086 variant that is heterologousto a N. meningitidis strain expressing B01. For example, in oneembodiment, the hSBA strain is an LP2086 (fHBP) subfamily B strain thatexpresses a lipoprotein 2086 variant that is heterologous to strainCDC1127. In a preferred embodiment, the hSBA strain is an LP2086 (fHBP)subfamily B strain that expresses a lipoprotein 2086 variant that isheterologous to strain CDC1573.

In one embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that is heterologous to a N.meningitidis strain expressing B01. In one embodiment, the immuneresponse is against N. meningitidis serogroup B B24 strain. In oneembodiment, the immune response is against N. meningitidis serogroup BB44 strain. In one embodiment, the immune response is against N.meningitidis serogroup B B16 strain. In one embodiment, the immuneresponse is against N. meningitidis serogroup B B03 strain. In oneembodiment, the immune response is against N. meningitidis serogroup BB09 strain. In one embodiment, the immune response is against N.meningitidis serogroup B B15 strain. In one embodiment, the immuneresponse is against N. meningitidis serogroup B B153 strain. In oneembodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that is heterologous to N.meningitidis strain CDC1573. In one embodiment, the immune response isbactericidal against a N. meningitidis serogroup B subfamily B strainthat expresses a factor H binding protein including an amino acidsequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the second polypeptide. In another embodiment, the immune response isbactericidal against a N. meningitidis serogroup B subfamily B strainthat expresses a factor H binding protein including an amino acidsequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identityto a factor H binding protein expressed by N. meningitidis strainCDC1573. In a preferred embodiment, the immune response is bactericidalagainst a N. meningitidis serogroup B subfamily B strain that expressesa factor H binding protein including an amino acid sequence that has atleast 80% identity, more preferably at least 87% identity, to a factor Hbinding protein expressed by N. meningitidis strain CDC1573. In anotherpreferred embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that expresses a factor Hbinding protein including an amino acid sequence that has 100% identityto a factor H binding protein expressed by N. meningitidis strainCDC1573.

In another embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that expresses a factor Hbinding protein including an amino acid sequence that has at most 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identity to the second polypeptide. Inanother embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that expresses a factor Hbinding protein including an amino acid sequence that has at most 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identity to a factor H binding proteinexpressed by N. meningitidis strain CDC1573. In a preferred embodiment,the immune response is bactericidal against a N. meningitidis serogroupB subfamily B strain that expresses a factor H binding protein includingan amino acid sequence that has at most 99% identity, more preferably atleast 88% identity, to a factor H binding protein expressed by N.meningitidis strain CDC1573. Any minimum value may be combined with anymaximum value described herein to define a range.

In one embodiment, the hSBA strain is an LP2086 (fHBP) B24 strain. Inanother embodiment, the hSBA strains is an LP2086 (fHBP) B44 strain. Ina further embodiment, the hSBA strains includes LP2086 (fHBP) B24 andLP2086 (fHBP) B44 strains. In one embodiment, the hSBA strains includesLP2086 (fHBP) A22, LP2086 (fHBP) A56, LP2086 (fHBP) B24, and LP2086(fHBP) B44 strains. In one embodiment, the hSBA strain includes B15. Inone embodiment, the hSBA strain includes B153. In another embodiment,the hSBA strain is an LP2086 B16 strain. In one embodiment, the hSBAstrain is an LP2086 B03 strain. In one embodiment, the hSBA strain is anLP2086 B09 strain. In a further embodiment, the hSBA strains includeB24, B16, B44, B03, and B09, or any combination thereof. In anotherembodiment, the hSBA strains include B24, B16, B44, A22, B03, B09, A12,A19, A05, and A07, or any combination thereof. For example, in oneembodiment, the hSBA strains include A22 and B24. In another embodiment,the hSBA strains include A06, A07, A12, A15, A19, A29, B03, B09, B15,and B16, or any combination thereof.

In one embodiment, the method induces an immune response against a N.meningitidis serogroup B subfamily A strain and against a N.meningitidis serogroup B subfamily B strain. Preferably, the immuneresponse is bactericidal against a N. meningitidis serogroup B subfamilyA strain and against a N. meningitidis serogroup B subfamily B strain.

In one embodiment, the immune response against the N. meningitidisserogroup B subfamily A strain is greater than the immune responseagainst the N. meningitidis serogroup B subfamily B strain. For example,in one embodiment, the immunogenic composition induces higherbactericidal titers against a N. meningitidis serogroup B subfamily Astrain than against a N. meningitidis serogroup B subfamily B strain,when tested under identical conditions. In one embodiment, the higherbactericidal titers against a N. meningitidis serogroup B subfamily Astrain occurs within 30 days after a second dose of the immunogeniccomposition against N. meningitidis. In one embodiment, the higherbactericidal titers against a N. meningitidis serogroup B subfamily Astrain occur in the absence of a third dose of the immunogeniccomposition against N. meningitidis.

In another embodiment, the immune response against the N. meningitidisserogroup B subfamily B strain is greater than the immune responseagainst the N. meningitidis serogroup B subfamily A strain. For example,in one embodiment, the immunogenic composition induces higherbactericidal titers against a N. meningitidis serogroup B subfamily Bstrain than against a N. meningitidis serogroup B subfamily A strain,when tested under identical conditions. In one embodiment, the higherbactericidal titers against a N. meningitidis serogroup B subfamily Bstrain occurs within 30 days after a second dose of the immunogeniccomposition against N. meningitidis. In one embodiment, the higherbactericidal titers against a N. meningitidis serogroup B subfamily Bstrain occur in the absence of a third dose of the immunogeniccomposition against N. meningitidis.

Titers

In one embodiment, the composition induces an increase in bactericidaltiter against N. meningitidis serogroup B in the human, as compared tothe bactericidal titer against N. meningitidis serogroup B in the humanprior to administration of a dose of the composition, when measuredunder identical conditions, e.g., in an hSBA. In one embodiment, theincrease in bactericidal titer is compared to the bactericidal titer inthe human before administration of the first dose of the composition, ascompared to the bactericidal titer in the human prior to administrationof the first dose of the composition, when measured under identicalconditions, e.g., in an hSBA. In one embodiment, the increase in titeris observed after a second dose of the composition, as compared to thebactericidal titer in the human prior to administration of the seconddose of the composition, when measured under identical conditions, e.g.,in an hSBA. In another embodiment, the increase in bactericidal titer isobserved after a third dose of the composition, as compared to thebactericidal titer in the human prior to administration of the thirddose of the composition, when measured under identical conditions, e.g.,in an hSBA.

In one embodiment, the composition induces a bactericidal titer againstN. meningitidis serogroup B in the human after administration of a dose,wherein the bactericidal titer is at least greater than 1-fold higherthan the bactericidal titer against N. meningitidis serogroup B in thehuman prior to administration of the dose, when measured under identicalconditions, e.g., in an hSBA. For example, the bactericidal titeragainst N. meningitidis serogroup B may be at least 1.01-fold, 1.1-fold,1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or 16-foldhigher in the human after receiving a dose of the composition, ascompared to the bactericidal titer against N. meningitidis serogroup Bin the human prior to administration of the dose, when measured underidentical conditions, e.g., in an hSBA.

In one embodiment, the composition when used in combination with anadditional immunogenic composition against human papillomavirus inducesan increase in virucidal titer against human papillomavirus in thehuman, as compared to the virucidal titer against human papillomavirusin the human prior to administration of a dose of the composition, whenmeasured under identical conditions. In one embodiment, the increase invirucidal titer is compared to the virucidal titer in the human beforeadministration of the first dose of the composition, as compared to thevirucidal titer in the human prior to administration of the first doseof the composition, when measured under identical conditions. In oneembodiment, the increase in titer is observed after a second dose of thecomposition, as compared to the virucidal titer in the human prior toadministration of the second dose of the composition, when measuredunder identical conditions. In another embodiment, the increase invirucidal titer is observed after a third dose of the composition, ascompared to the virucidal titer in the human prior to administration ofthe third dose of the composition, when measured under identicalconditions.

In one embodiment, the composition when used in combination with anadditional immunogenic composition against human papillomavirus inducesa virucidal titer in the human after administration of a dose, whereinthe virucidal titer against human papillomavirus is at least greaterthan 1-fold higher than the virucidal titer against human papillomavirusin the human prior to administration of the dose, when measured underidentical conditions. For example, the virucidal titer against humanpapillomavirus may be at least 1.01-fold, 1.1-fold, 1.5-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or 16-fold higher in thehuman after receiving a dose of the composition, as compared to thevirucidal titer against human papillomavirus in the human prior toadministration of the dose, when measured under identical conditions.

In one embodiment, a “responder” refers to a human, wherein thecomposition induces a bactericidal titer against N. meningitidisserogroup B in the human after administration of a dose, wherein thebactericidal titer against N. meningitidis serogroup B is at leastgreater than 1-fold higher than the bactericidal titer against N.meningitidis serogroup B in the human prior to administration of thedose. In a preferred embodiment, the responder achieves at least a4-fold rise in hSBA titer, as compared to a bactericidal titer in thehuman prior to administration of the dose. Such a responder may bereferred to as having a protective titer.

In one embodiment, the hSBA titer is the reciprocal of the highestdilution of a serum sample that produces a measurable effect. Forexample, in one embodiment, the hSBA titer is the reciprocal of thehighest 2-fold dilution of a test serum that results in at least a 50%reduction of MnB bacteria (50% bacterial survival) compared to the T30CFU value (i.e., the number of bacteria surviving after incubation inassay wells containing all assay components except test serum; 100%bacterial survival).

In one embodiment, the composition induces a bactericidal titer againstN. meningitidis serogroup B in the human after receiving the first dosethat is at least 2-fold higher than the bactericidal titer against N.meningitidis serogroup B in the human prior to receiving the first dose(e.g., higher than the bactericidal titer in the human in the absence ofthe first dose), when measured under identical conditions in the hSBA.In one embodiment, the composition induces a bactericidal titer againstN. meningitidis serogroup B in the human that is at least 4-fold higherthan the bactericidal titer against N. meningitidis serogroup B in thehuman prior to receiving the first dose, when measured under identicalconditions in a human serum bactericidal assay that utilizes humancomplement (hSBA). In one embodiment, the composition induces abactericidal titer against N. meningitidis serogroup B in the human thatis at least 8-fold higher than the bactericidal titer against N.meningitidis serogroup B in the human prior to receiving the first dose,when measured under identical conditions in a human serum bactericidalassay that utilizes human complement (hSBA).

In a preferred embodiment, the human serum complement is derived from ahuman having low intrinsic bactericidal activity for a given SBA teststrain. Low intrinsic bactericidal activity refers to, for example, abactericidal titer that is at least less than a 1:4 dilution against thegiven SBA test strain. In one embodiment, the human complement isderived from a human having an hSBA titer that is at least less than1:4, such as a 1:2 dilution, against the given SBA test strain, whereinthe composition was not administered to the human.

A human may exhibit an hSBA titer of less than 1:4 prior toadministration of a composition, such as the bivalent rLP2086composition, or a human may exhibit an hSBA titer of ≥1:4 prior toadministration of the composition. Accordingly, in preferred embodimentsand examples, administration of at least one dose of the composition tothe human results in an hSBA titer that is at least greater than 1:4,such as, for example, an hSBA titer of ≥1:8, an hSBA titer of ≥1:16, andan hSBA titer of ≥1:32. The respective Examples described herein includeassessments of the proportion of human subjects having an hSBA titer≥1:8 and/or ≥1:16, wherein the bivalent rLP2086 composition wasadministered to the human. Such preferred assessments of hSBA titersgreater than 1:4 show that the protection, i.e., the bactericidal immuneresponse induced in the human, is associated with the composition.

In one embodiment, the human has an hSBA titer equal to or greater thanthe hSBA's lower limit of quantitation (LLOQ) after administration ofthe first dose of the composition. In another embodiment, the human hasan hSBA titer equal to or greater than the hSBA's LLOQ afteradministration of the second dose of the composition. In anotherembodiment, the human has an hSBA titer equal to or greater than thehSBA's LLOQ after administration of the third dose of the composition.

Additional Immunogenic Compositions

In one aspect, the invention relates to a method of inducing abactericidal immune response against a N. meningitidis serogroup Bsubfamily A strain, and against a N. meningitidis serogroup B subfamilyB strain in a human, in addition to inducing an immune response againstat least one of any of the following: a N. meningitidis serogroup Astrain, a N. meningitidis serogroup C strain, a N. meningitidisserogroup Y strain, and/or a N. meningitidis serogroup W135 strain;diphtheria, tetanus, and/or pertussis; hepatitis A virus; humanpapillomavirus; diphtheria, tetanus, pertussis and/or poliomyelitis; orany combination thereof.

The method includes administering to the human an effective amount ofthe immunogenic composition against N. meningitidis serogroup B, andconcomitantly administering to the human at least one of the followingimmunogenic compositions: (i) an immunogenic composition against a N.meningitidis serogroup A strain, a N. meningitidis serogroup C strain, aN. meningitidis serogroup Y strain, and/or a N. meningitidis serogroupW135 strain; (ii) an immunogenic composition against diphtheria,tetanus, and pertussis; (iii) an immunogenic composition againsthepatitis A virus; (iv) an immunogenic composition against humanpapillomavirus; (v) an immunogenic composition against diphtheria,tetanus, pertussis and poliomyelitis; or (vi) any combination thereof.

In one embodiment, the method includes administering to the human aneffective amount of the immunogenic composition against N. meningitidisserogroup B, and concomitantly administering to the human at least oneof the following immunogenic compositions: (i) MENACTRA, MENVEO, orNIMENRIX; (ii) ADACEL; (iii) HAVRIX; (iv) GARDASIL; (v) REPEVAX; or (vi)any combination thereof.

For example, in exemplary embodiments, the method includes concomitantlyadministering to the human at least one of the following combination ofcompositions:

Exemplary The rLP2086 MCV4 Tdap HAV HPV dTaP composition: compositioncomposition composition composition composition composition Combination1 rLP2086 MCV4 Tdap HAV HPV dTaP composition composition compositioncomposition composition composition Combination 2 rLP2086 Tdap HAV HPVdTaP composition composition composition composition compositionCombination 3 rLP2086 MCV4 HAV HPV dTaP composition compositioncomposition composition composition Combination 4 rLP2086 MCV4 Tdap HPVdTaP composition composition composition composition compositionCombination 5 rLP2086 MCV4 Tdap HAV dTaP composition compositioncomposition composition composition Combination 6 rLP2086 MCV4 Tdap HAVHPV composition composition composition composition compositionCombination 7 rLP2086 MCV4 Tdap HAV composition composition compositioncomposition Combination 8 rLP2086 MCV4 HAV HPV composition compositioncomposition composition Combination 9 rLP2086 MCV4 HPV dTaP compositioncomposition composition composition Combination 10 rLP2086 MCV4 TdapdTaP composition composition composition composition Combination 11rLP2086 Tdap HAV HPV composition composition composition compositionCombination 12 rLP2086 Tdap HPV dTaP composition composition compositioncomposition Combination 13 rLP2086 Tdap HAV dTaP composition compositioncomposition composition Combination 14 rLP2086 HAV HPV dTaP compositioncomposition composition composition Combination 15 rLP2086 MCV4 HAV dTaPcomposition composition composition composition Combination 16 rLP2086MCV4 Tdap HPV composition composition composition compositionCombination 17 rLP2086 MCV4 Tdap composition composition compositionCombination 18 rLP2086 MCV4 HAV composition composition compositionCombination 19 rLP2086 MCV4 HPV composition composition compositionCombination 20 rLP2086 MCV4 dTaP composition composition compositionCombination 21 rLP2086 Tdap HAV composition composition compositionCombination 22 rLP2086 Tdap HPV composition composition compositionCombination 23 rLP2086 Tdap dTaP composition composition compositionCombination 24 rLP2086 HAV HPV composition composition compositionCombination 25 rLP2086 HAV dTaP composition composition compositionCombination 26 rLP2086 HPV dTaP composition composition compositionCombination 27 rLP2086 MCV4 composition composition Combination 28rLP2086 Tdap composition composition Combination 29 rLP2086 HAVcomposition composition Combination 30 rLP2086 HPV compositioncomposition Combination 31 rLP2086 dTaP composition compositionCombination 32 rLP2086 MENACTRA, composition MENVEO, or NIMENRIXCombination 33 rLP2086 ADACEL composition Combination 34 rLP2086 HAVRIXcomposition Combination 35 rLP2086 GARDASIL composition Combination 36rLP2086 REPEVAX composition

“Concomitant” and “concomitantly” and “coadministered” and“coadministration” as used herein refer to the administration of atleast two compositions to a human simultaneously or within a time periodduring which the effects of the first administered composition is stilleffective in the human. If the first composition is, e.g., the rLP2086composition and the second composition is an MCV4 composition, theconcomitant administration of the second composition can occur withinone to seven days, preferably within 48 hours, more preferably within 24hours, of the administration of the first composition. In oneembodiment, the concomitant administration occurs within at most 1 hour,2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours,10 hours, 11 hours, 12 hours, 24 hours, or 48 hours of theadministration of the first composition. In another embodiment, theconcomitant administration occurs simultaneously to administration ofthe first composition.

In a concomitant administration, the first composition may beadministered before, after, or simultaneously to the additionalcomposition(s) within the specified time period. For example, in oneembodiment, the first composition, e.g., the rLP2086 composition, isadministered to the human first and the second composition, e.g., anMCV4 composition, is administered within 24 hours after the rLP2086composition. In another embodiment, the first composition, e.g., an MCV4composition, is administered to the human first and the secondcomposition, e.g., the rLP2086 composition, is administered to the humanwithin 24 hours after the MCV4 composition.

In one embodiment, the additional composition(s) is administered before,after, or simultaneously to the first dose of the rLP2086 compositionwithin the specified time period. In another embodiment, In oneembodiment, the additional composition(s) is administered before, after,or simultaneously to the second dose of the rLP2086 composition withinthe specified time period. In a further embodiment, the additionalcomposition(s) is administered before, after, or simultaneously to thethird dose of the rLP2086 composition within the specified time period.In yet another embodiment, the additional composition(s) is administeredbefore, after, or simultaneously to a booster dose of the rLP2086composition within the specified time period.

The inventors surprisingly discovered that the rLP2086 composition maybe administered with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,or 14 additional antigens without negatively affecting the immuneresponse against N. meningitidis. For example, Example 11 demonstratesthat substantial hSBA responses to N. meningitidis strains and to eachof the at least 10 MCV4 and Tdap antigens were observed.

Example 13 demonstrates that the rLP2086 composition may becoadministered with the 14 antigens included amongst HPV4, MCV4, andTdap vaccines while inducing an effective immune response, which mayinclude a bactericidal and/or virucidal immune response.

Accordingly, in one aspect, the invention relates to a method ofinducing an immune response against a N. meningitidis serogroup Astrain, a N. meningitidis serogroup C strain, a N. meningitidisserogroup Y strain, a N. meningitidis serogroup W135 strain, and a N.meningitidis serogroup B strain. In a preferred embodiment, the immuneresponse comprises a bactericidal immune response. The method includesadministering to the human an effective amount of a N. meningitidisrLP2086 composition, said composition including a) a first lipidatedpolypeptide having the amino acid sequence set forth in SEQ ID NO: 1,and b) a second lipidated polypeptide having the amino acid sequence setforth in SEQ ID NO: 2; and a further composition including a mixture offour distinct and separately made protein-capsular polysaccharideconjugates, wherein the first conjugate includes a purified N.meningitidis capsular polysaccharide of serogroup W-135 conjugated to acarrier protein, the second conjugate includes a purified N.meningitidis capsular polysaccharide of serogroup Y conjugated to acarrier protein, the third conjugate includes a purified N. meningitidiscapsular polysaccharide of serogroup A conjugated to a purified carrierprotein, and the fourth conjugate includes a purified N. meningitidiscapsular polysaccharide of serogroup C conjugated to a carrier protein,wherein the carrier protein is selected from the group consisting ofdiphtheria toxoid, CRM₁₉₇, and tetanus toxoid.

In one preferred embodiment, the method includes administering an MCV4composition, wherein the carrier protein is diphtheria toxoid. In oneembodiment, the method includes administering MENACTRA vaccine.

In another embodiment, the method includes administering an MCV4composition wherein the carrier protein is CRM₁₉₇. In one embodiment,the method includes administering MENVEO vaccine. MENVEO [Meningococcal(Groups A, C, Y and W-135) Oligosaccharide Diphtheria CRM197 ConjugateVaccine] is a sterile liquid vaccine administered by intramuscularinjection that contains N. meningitidis serogroup A, C, Y and W-135oligosaccharides conjugated individually to Corynebacterium diphtheriaeCRM197 protein.

In yet another embodiment, the method includes administering an MCV4composition wherein the carrier protein is tetanus toxoid. In oneembodiment, the method includes administering NIMENRIX vaccine.NIMENRIX® (meningococcal polysaccharide groups A, C, W-135 and Yconjugate vaccine) is a tetravalent meningococcal polysaccharideconjugated vaccine including N. meningitidis capsular polysaccharides A,C, W-135 and Y each coupled to tetanus toxoid as a carrier protein. TheN. meningitidis serogroups A and C polysaccharides are conjugated withan adipic dihydrazide (AH) spacer and indirectly conjugated to thetetanus toxoid whereas the W-135 and Y polysaccharides are conjugateddirectly to tetanus toxoid.

In one embodiment, the method increases titers against N. meningitidisserogroup A, C, W-135, and/or Y at least greater than 1-fold, at least2-fold, at least 3-fold, at least 4-fold, or more, as compared to thetiters in the human prior to administration of the immunogeniccompositions. In another embodiment, the method increases titers againstN. meningitidis serogroup A, C, W-135, and Y at least greater than1-fold, at least 2-fold, at least 3-fold, at least 4-fold, or more, ascompared to the titers in the human prior to administration of theimmunogenic compositions. In another embodiment, the method increasestiters against N. meningitidis serogroup A, C, W-135, and Y andserogroup B at least greater than 1-fold, at least 2-fold, at least3-fold, at least 4-fold, or more, as compared to the titers in the humanprior to administration of the immunogenic compositions.

In another aspect, the invention relates to a method of inducing animmune response against a N. meningitidis serogroup B strain and againsttetanus, diphtheria, and pertussis in a human. In a preferredembodiment, the immune response comprises a bactericidal immuneresponse. In another preferred embodiment, the immune response comprisesa virucidal immune response. In another preferred embodiment, the immuneresponse comprises a bactericidal and virucidal immune response. Themethod includes administering to the human an effective amount of a N.meningitidis rLP2086 composition, said composition including a) a firstlipidated polypeptide having the amino acid sequence set forth in SEQ IDNO: 1, and b) a second lipidated polypeptide having the amino acidsequence set forth in SEQ ID NO: 2; and a further composition includingtetanus and diphtheria toxoids and pertussis antigens, preferablyadsorbed on aluminum phosphate. In one embodiment, the method includesadministering ADACEL vaccine.

In one embodiment, the method increases titers against tetanus,diphtheria, and and/or pertussis at least greater than 1-fold, at least2-fold, at least 3-fold, at least 4-fold, or more, as compared to thetiters in the human prior to administration of the immunogeniccompositions. In another embodiment, the method increases titers againsttetanus, diphtheria, and pertussis at least greater than 1-fold, atleast 2-fold, at least 3-fold, at least 4-fold, or more, as compared tothe titers in the human prior to administration of the immunogeniccompositions. In another embodiment, the method increases titers againsttetanus, diphtheria, and pertussis, and N. meningitidis serogroups A, C,W-135, and Y and serogroup B at least greater than 1-fold, at least2-fold, at least 3-fold, at least 4-fold, or more, as compared to thetiters in the human prior to administration of the immunogeniccompositions.

In one aspect, the invention relates to a method of inducing an immuneresponse against a N. meningitidis serogroup B strain, against N.meningitidis serogroup A, C, Y, and W135 strains, and against tetanus,diphtheria, and pertussis in a human. In a preferred embodiment, theimmune response comprises a bactericidal immune response. In anotherpreferred embodiment, the immune response comprises a virucidal immuneresponse. In another preferred embodiment, the immune response comprisesa bactericidal and virucidal immune response. In one embodiment, themethod includes concomitantly administering the rLP2086 composition,MENACTRA, and ADACEL vaccines.

In one aspect, the invention relates to a method of inducing an immuneresponse against a N. meningitidis serogroup B strain, against N.meningitidis serogroups A, C, Y, and W135 strains, and against tetanus,diphtheria, and pertussis, and further against against humanpapillomavirus in a human. In a preferred embodiment, the immuneresponse comprises a bactericidal immune response. In another preferredembodiment, the immune response comprises a virucidal immune response.In another preferred embodiment, the immune response comprises abactericidal and virucidal immune response. In one embodiment, themethod includes concomitantly administering the rLP2086 composition,MENACTRA, and ADACEL vaccines. In one embodiment, the method includesconcomitantly administering the rLP2086 composition, MENACTRA, ADACEL,and GARDASIL vaccines.

The inventors surprisingly discovered that the immunogenic compositionagainst N. meningitidis may be administered with an immunogeniccomposition against human papillomavirus (HPV) without negativelyaffecting the bactericidal response against N. meningitidis. Asexplained in Example 7 and Example 8, substantial hSBA responses to N.meningitidis test strains were observed among humans who wereadministered with the immunogenic composition against N. meningitidisand GARDASIL and in humans who were administered with the immunogeniccomposition against N. meningitidis and saline. Additional increases inhSBA responses were observed about 1 month after a third dose of theimmunogenic composition against N. meningitidis.

Moreover, the inventors surprisingly discovered that robust immuneresponses against both N. meningitidis and HPV were generated in thehuman following an administration of both the immunogenic compositionagainst N. meningitidis and the immunogenic composition against HPV, ascompared to the immune response in the human before administration ofthe compositions. As explained in Example 7 and Example 8, titersagainst HPV increased in the human after an administration of theimmunogenic composition against N. meningitidis and GARDASIL, ascompared to the titers in the human prior to administration of theimmunogenic compositions. The increase in titers against HPV was atleast greater than 1-fold, at least 2-fold, at least 3-fold, at least4-fold, or more.

Accordingly, in one embodiment, the method includes inducing an immuneresponse against N. meningitidis in a human, wherein the method furtherincludes administering to the human an immunogenic composition againsthuman papillomavirus. Preferably, the immune response is bactericidalagainst N. meningitidis. In one embodiment, the method further includesinducing an immune response against HPV. In a preferred embodiment, themethod further includes inducing an immune response against at least oneof human papillomavirus types 6, 11, 16, and 18, or any combinationthereof. In one embodiment, the immunogenic composition against HPV isadministered to the human within 24 hours of administering saidcomposition against N. meningitidis.

In one embodiment, the method includes inducing an immune responseagainst N. meningitidis in a human, wherein the method further includesadministering to the human an immunogenic composition against HPV.Preferably, the immune response is bactericidal against N. meningitidis.In one embodiment, the method further includes inducing an immuneresponse against HPV. In a preferred embodiment, the method furtherincludes inducing an immune response against at least one of humanpapillomavirus types 6, 11, 16, and 18, or any combination thereof. Inone embodiment, the immunogenic composition against human papillomavirusis administered to the human within 24 hours of administering saidcomposition against N. meningitidis.

In another aspect, the inventors surprisingly discovered that theimmunogenic composition against N. meningitidis may be administered withan immunogenic composition against diphtheria, tetanus, acellularpertussis, and inactivated poliomyelitis virus (dTaP) without negativelyaffecting the bactericidal response against N. meningitidis. Asexplained in Example 4, substantial hSBA responses to N. meningitidistest strains were observed among humans who were administered with theimmunogenic composition against N. meningitidis and REPEVAX. Additionalincreases in hSBA responses were observed about 1 month after a thirddose of the immunogenic composition against N. meningitidis.

Moreover, the inventors surprisingly discovered that robust immuneresponses against both N. meningitidis and dTaP were generated in thehuman following an administration of both the immunogenic compositionagainst N. meningitidis and the immunogenic composition against dTaP, ascompared to the immune response in the human before administration ofthe compositions. As explained in Example 4, titers against dTaPincreased in the humanafter an administration of the immunogeniccomposition against N. meningitidis and REPEVAX, as compared to thetiters in the human prior to administration of the immunogeniccompositions. The increase in titers against dTaP was at least greaterthan 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, or more.

Methods and Administration

In one aspect, the invention relates to a method of inducing an immuneresponse in a human. In a preferred embodiment, the immune responsecomprises a bactericidal immune response. In another preferredembodiment, the immune response comprises a virucidal immune response.In another preferred embodiment, the immune response comprises abactericidal and virucidal immune response. In another preferredembodiment, the immune response comprises inactivating the bacteriaand/or virus.

In another aspect, the invention relates to a method of vaccinating ahuman. In one embodiment, the method includes administering to the humanat least one dose of the composition described above. In anotherembodiment, the method includes administering to the human at least afirst dose and a second dose of the composition described above.

Surprisingly, the inventors discovered that a two-dose schedule of therLP2086 composition induced a bactericidal titer against diverseheterologous subfamily A and against diverse heterologous subfamily Bstrains in the human. For example, the percentage of humans with an hSBAtiter ≥1:8 was 90% or greater for SBA test strains expressing LP2086(fHBP) A22 or LP2086 (fHBP) A56 following a two-dose schedule of thecomposition described above. See Example 1.

In one embodiment, the second dose is administered at least 20, 30, 50,60, 100, 120, 160, 170, or 180 days after the first dose, and at most250, 210, 200, or 190 days after the first dose. Any minimum value maybe combined with any maximum value described herein to define a range.

In another embodiment, the second dose is administered about 30 daysafter the first dose. In another embodiment, the second dose isadministered about 60 days after the first dose, such as, for example,in a 0, 2 month immunization schedule. In another embodiment, the seconddose is administered about 180 days after the first dose, such as, forexample, in a 0, 6 month immunization schedule. In yet anotherembodiment, the second dose is administered about 120 days after thefirst dose, such as, for example, in a 2, 6 month immunization schedule.

In one embodiment, the method includes administering to the human twodoses of the composition and at most two doses. In one embodiment, thetwo doses are administered within a period of about 6 months after thefirst dose. In one embodiment, the method does not include furtheradministration of a booster to the human. A “booster” as used hereinrefers to an additional administration of the composition to the human.Administering to the human at most two doses of the composition may beadvantageous. Such advantages include, for example, facilitating a humanto comply with a complete administration schedule and facilitatingcost-effectiveness of the schedule.

In one embodiment, the first dose and the second dose are administeredto the human over a period of about 25, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 days, and most 400,390, 380, 370, 365, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260,250, 240, 230, 220, 210, or 200 days after the first dose. Any minimumvalue may be combined with any maximum value described herein to definea range.

In one embodiment, the first dose and the second dose are administeredto the human over a period of about 30 days. In another embodiment, thefirst dose and the second dose are administered to the human over aperiod of about 60 days. In another embodiment, the first dose and thesecond dose are administered to the human over a period of about 180days.

Three Doses

The inventors further surprisingly discovered that a three-dose scheduleof the rLP2086 composition induced a broader bactericidal titer againststrains expressing heterologous LP2086 (fHBP) subfamily B strains in agreater percentage of humans than a two-dose schedule. For example, thepercentage of humans with a hSBA titer ≥1:8 was 65% or greater for SBAtest strains LP2086 (fHBP) B24 and LP2086 (fHBP) B44 following atwo-dose schedule of the composition described above. The percentage ofhumans with a hSBA titer ≥1:8 was 86% or greater for SBA test strainsB24 and B44 following a three-dose schedule of the composition describedabove. See Example 1.

Accordingly, in one embodiment, a three-dose schedule of the compositioninduces a bactericidal titer against multiple strains expressing LP2086(fHBP) heterologous to the first and/or second polypeptide in a greaterpercentage of humans than a two-dose schedule.

In one embodiment, the method includes administering to the human threedoses of the composition. In another embodiment, the method includesadministering at most three doses of the composition. In one embodiment,the three doses are administered within a period of about 6 months afterthe first dose. In one embodiment, the method includes an administrationof a booster dose to the human after the third dose. In anotherembodiment, the method does not include administration of a booster doseto the human after the third dose. In another embodiment, the methoddoes not further include administering a fourth or booster dose of thecomposition to the human. In a further embodiment, at most three doseswithin a period of about 6 months are administered to the human.

In an exemplary embodiment, the second dose is administered about 30days after the first dose, and the third dose is administered about 150days after the second dose, such as, for example, in a 0, 1, 6 monthimmunization schedule. In another exemplary embodiment, the second doseis administered about 60 days after the first dose, and the third doseis administered about 120 days after the second dose, such as, forexample, in a 0, 2, 6 month immunization schedule.

In one embodiment, the first dose, second dose, and third dose areadministered to the human over a period of about 150, 160, 170, or 180days, and at most 240, 210 200, or 190 days. Any minimum value may becombined with any maximum value described herein to define a range.Preferably, the first dose, second dose, and third dose is administeredto the human over a period of about 180 days or 6 months. For example,the second dose may be administered to the human about 60 days after thefirst dose, and the third dose may be administered to the human about120 days after the second dose. Accordingly, an exemplary schedule ofadministration includes administering a dose to the human at aboutmonths 0, 2, and 6.

As described above, multiple doses of the immunogenic composition may beadministered to the human, and the number of days between each dose mayvary. An advantage of the method includes, for example, flexibility fora human to comply with the administration schedules.

EXAMPLES

The following Examples illustrate embodiments of the invention. Unlessnoted otherwise herein, reference is made in the following Examples toan investigational bivalent recombinant vaccine (rLP2086), which is apreferred exemplary embodiment of a composition including 60 μg of afirst lipidated polypeptide including the amino acid sequence set forthin SEQ ID NO: 1 per 0.5 mL dose, 60 μg of a second lipidated polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 2 per 0.5 mLdose, 2.8 molar ratio polysorbate-80 to the first polypeptide, 2.8 molarratio polysorbate-80 to the second polypeptide, 0.5 mg Al³⁺/ml of thecomposition, 10 mM histidine, and 150 mM sodium chloride. Morespecifically, the investigational bivalent recombinant rLP2086 vaccineincludes (a) 60 μg of a first lipidated polypeptide including the aminoacid sequence set forth in SEQ ID NO: 1; (b) 60 μg of a second lipidatedpolypeptide including the amino acid sequence set forth in SEQ ID NO: 2;(c) 18 μg polysorbate-80; (d) 250 μg aluminum; (e) 780 μg histidine, and(f) 4380 μg sodium chloride. Each dose was 0.5 mL.

Example 1: Safety, Tolerability, and Immunogenicity of anInvestigational Meningococcal Serogroup B Bivalent (MnB) rLP2086 Vaccinein Healthy Adolescents when Administered in Regimens of 2 or 3 Doses inHealthy Subjects Aged 11 to 18 Years Background:

Safety, tolerability, and immunogenicity of an investigational bivalent,recombinant vaccine (rLP2086) were studied in healthy adolescents 11-18years of age using 5 dose regimens including 2 or 3 vaccinations (Table1).

The vaccine is a 0.5 ml-dose formulated to contain 60 μg each of apurified subfamily A and a purified subfamily B rLP2086 protein, 2.8molar ratio polysorbate-80, and 0.25 mg of Al³⁺ as AlPO₄, 10 mMhistidine-buffered saline at pH 6.0.

Saline is used as a placebo because there is no known proven safe,immunogenic, and effective vaccine against MnB that could serve as anactive control. The normal saline solution includes 0.9% sodium chloridein a 0.5 ml dose.

Methods:

All subjects in this phase 2, randomized, placebo-controlled,single-blind study attended vaccination visits at months 0, 1, 2 and 6.For blinding, a saline control was given when vaccine was not scheduled.Serum bactericidal assays using human complement (hSBA) were performedwith 4 MnB test strains expressing LP2086 (fHBP) fHBP variants A22, A56,B24 and B44 (i.e., the 4 “primary hSBA test strains” in the primaryendpoint analysis), all of which are different from the variants in thevaccine. Unsolicited adverse events (AE), solicited local and systemicreactions, and antipyretic use were assessed.

Geometric mean hSBA titers were computed for each primary strain at eachblood sampling time point along with 2-sided 95% confidence intervals(CIs). Geometric mean fold rises were computed along with 95% CIs.

A responder was defined as a subject with an hSBA titer equal or abovethe lower limit of quantitation (LLOQ) of the hSBA assays. The LLOQ foreach of the 4 hSBA test strains in the primary endpoint analysis was anhSBA titer equal to 1:8. The limit of detection (LOD) for each primarytest strain was a titer equal to 1:4 (widely viewed as the correlate ofprotection against meningococcal disease).

Results:

1 month after the last vaccine dose, 86-99% subjects (after 3 doses;P<0.001) and 69-100% of subjects (after 2 doses) had hSBA titers to eachMnB test strain. After study dose 1, 19-27% (1.1-4.3% severe) and 23-27%(0.0-1.0% severe) of rLP2086 recipients experienced redness andswelling, respectively, by group. Injection site pain was the mostcommon local reaction after study dose 1 (7.6-13.1% severe). Fever ≥38°C. after the first study dose of the bivalent rLP2086 vaccine wasexperienced in 3.3-6.5% by group compared to 2.1% in saline recipients.Local and systemic reactions were generally more frequent after dose 1than after subsequent doses. 43 of 1712 subjects (2.5%) reported 51serious AEs; 2 cases were considered related (1 case of vertigo, chillsand headache and 1 case of fever and vomiting). No deaths were reported.

TABLE 1 Table. Statistical Analysis on Proportion of Evaluable StudySubjects Achieving hSBA Titer ≥8 for Each Primary Strain 1 Month AfterLast Dose of Bivalent rLP2086 - Evaluable immunogenicity PopulationGroup 1 Group 2 Group 3 Group 4 Group 5 Strain (0, 1, 6 mo) (0, 2, 6 mo)(0, 6 mo) (0, 2 mo) (2, 6 mo) [variant] n⁺/N

%(95% Cl)

n

/N

% (95%. Cl)

n

/N

% (95% Cl)

n

/N

%(95% Cl)

n⁺/N^(±) % (95% Cl)

PMB80 330/360 91.7

, 339/357 95.0⁵ 345/369 93.5

218/230 90.8 102/111 91.9 [A22] (88.3, 94.3) (92.1, 97.01) (90.5, 95.8)(96.3, 94.1) (95.2, 96.2) PMB2001 360/362 99.4

  355/359 98.9

364/370 98.4

240/240 100.0 112/113 99.1 [A56] (98.0, 99.9) (97.2, 99.7) (98.5, 99.4)(98.5, 100.0) (95.2, 100.0) PMB2948 315/354 89.0

  313/354 88.4

291/359 81.

  173/237 73.0  76/110 69.1 [B24] (85.2, 92.0) (84.6, 91.6) (76.6, 85.0)(66.9, 78.5) (59.6, 77.6) PMB2707 315/356 88.5

  303/352 80.1

276/350 77.5

164/234 70.1 81/111 73.0 [B44] (84.7, 91.6) (82.0, 89.5) (72.2, 82.3)(63.8, 75.9) (63.7, 81.0)

Lower limit of quantification for all strains = 8

Number of subjects with hSBA fiter ≥ 8.

Number of subject with valid hSBA fiters.

P < 0.001 using one-sided exact test based on binomial,distribution,values

0.0125 are considered significant

Exact 2-sided confidence interval (Clopper and Pearson) based, upon theobserved proportion of subjects.

indicates data missing or illegible when filedConclusions: Bivalent rLP2086 had an acceptable safety profile. All 5dosing regimens yielded hSBA titers against all 4 test strains in a highproportion of subjects. The higher proportions against some test strainsafter 3 doses compared with 2 doses indicate that 3 doses may providethe broadest protection against diverse MnB clinical strains. Globalphase 3 clinical trials are underway with the bivalent rLP2086 vaccine.

One of the objectives of this study was to assess the immune response,as measured by hSBA performed with MnB strains expressing LP2086subfamily A and B proteins, 1 month after the third vaccination withbivalent rLP2086, among Group 1 subjects (0-, 1-, and 6-month scheduleas randomized) and among Group 2 subjects (0-, 2-, and 6-month scheduleas randomized). An endpoint for the immunogenicity analysis was theproportion of subjects in Groups 1 and 2 achieving an hSBA titer LLOQ atMonth 7 (or 1 month after the third dose of bivalent rLP2086) for eachof the 4 primary MnB test strains (A22, A56, B24, and B44). The LLOQ was1:8 for the 4 primary MnB test strains.

For the evaluable immunogenicity population, the proportion of subjectsin Group 1 achieving an hSBA titer ≥1:8 after 3 doses of bivalentrLP2086 was 91.7% for A22, 99.4% for A56, 89% for B24, and 88.5% for B44(See Table 1 above). Since the lower limit of the 97.5% CI was >50% forall strains (87.8%, p<0.001, 97.8%, p<0.001, 84.7%, p<0.001; and 84.1%,p<0.001 for strains A22, A56, B24, and B44, respectively, the studyobjective was met for subjects in Group 1.

For Group 2, the proportion of subjects achieving an hSBA titer ≥1:8after 3 doses of bivalent rLP2086 was 95.0% for A22, 98.9% for A56,88.4% for B24, and 86.1% for B44 (See Table 1 above). Similar to whatwas seen for Group 1, the lower limit of the 97.5% CI was >50% for allstrains (91.7%, p<0.001; 96.9%, p<0.001; 84.1%, p<0.001; and 81.4%,p<0.001 for strains A22, A56, B24, and B44, respectively, demonstratingthat the objective was also met for the subjects in Group 2.

A secondary objective was to assess the immune response, as measured byhSBA performed with MnB strains expressing LP2086 subfamily A and Bproteins, 1 month after the second dose of bivalent rLP2086, among group3 subjects (0- and 6-month schedule as randomized). This secondaryobjective was the proportion of subjects in Group 3 achieving an hSBAtiter ≥LLOQ (1:8) at Month 7 (or 1 month after the second dose ofbivalent rLP2086) for each of the 4 primary MnB test strains.

This secondary objective was also met since the proportion of subjectsin Group 3 achieving an hSBA titer ≥1:8 after 2 doses of bivalentrLP2086 was 93.5%, 98.4%, 81.1%, and 77.5% for the primary MnB teststrains with the lower limit of the 97.5% CI>50% for all strains (90.0%,p<0.001; 96.2%, p<0.001; 76.0%, p<0.001; and 72.2%, p<0.001 for strainsA22, A56, B24, and B44, respectively. See Table 1 above).

Another secondary objective was the proportion of subjects with hSBAtiter LLOQ for each of the 4 primary MnB test strains at each bloodsampling time point for subjects in Groups 1 to 5. The LLOQ for each ofthe 4 primary hSBA test strains was a titer of ≥1:8. The proportions ofsubjects with an hSBA titer ≥1:8 by study time for the evaluableimmunogenicity population is shown in Table 1 above.

The proportion of subjects who had an hSBA titer ≥1:8 after 1 dose ofbivalent rLP2086 (Group 5 [2- and 6-month schedule] 1 month afterInjection 3) was 55.9% for A22, 67.6% for A56, 56.9% for B24, and 23.8%for B44.

The proportion of subjects who had an hSBA titer ≥1:8 one month after 2doses of bivalent rLP2086 ranged from 74.6% to 100% for subfamily Astrains, and from 54.0% to 81.1% for subfamily B strains. After 3 doses,the proportion increased and ranged from 91.7% to 99.4% and from 86.1%to 89.0% for subfamily A and B strains, respectively.

Example 2: Serum Bactericidal Assay Using Human Complement (HSBA)

MnB clearance from the human bloodstream is primarily achieved bycomplement-mediated bacteriolysis and an intact complement system isimportant for resistance against infections caused by MnB. The in vivocomplement-mediated bacteriolysis of MnB is mimicked in vitro by theserum bactericidal assay using human complement (hSBA), a functionalserological assay shown to be the surrogate of protection formeningococcal disease. That is, demonstration of bacterial killing inthe serum bactericidal assay using human complement (hSBA) correlateswith protection against meningococcal disease. Immunity elicited by thevaccine is determined using hSBAs against 4 MnB strains (fHBP variantsA22, A56, B24, and B44).

The four primary MnB test strains were used in the hSBAs described inthe Examples for the determination of endpoints. That is, these strainswere used to estimate vaccine efficacy using hSBA immunogenicityendpoints. These test strains represent 4 of the 6 fHBP phylogeneticsubgroups that account for >90% of disease isolates circulating in theUSA and Europe.

On 29 Oct. 2014, TRUMENBA® (bivalent recombinant lipoprotein 2086[rLP2086]) was approved in the United States to prevent invasivemeningococcal disease caused by MnB in individuals aged 10 to 25 years.Approval of TRUMENBA® was based on demonstration of immune response, asmeasured by serum bactericidal activity against four serogroup B strainsrepresentative of IMD strains in the United States and Europe.

Identity to Lipo- matched oligo- fHBP saccharides subfamily Sialationvaccine fHBP Level Variant component subgroup CC PorA (mol %) A56 98.1%N1C2 CC213 P1.22, 14 55% B44 91.6% N4/N5 CC269 P1.19- 23% 1, 10-4 A2288.9% N202 CC41/44 P1.21, 16 84% B24 86.2% N6 CC32 P1.12- 22% 1, 13-1

In selecting the 4 primary MnB test strains from invasive diseaseisolates, an approach was used which took into account the populationdistribution of the in vitro LP2086 surface expression. Furthermore, thehSBA test strains had to show low baseline hSBA positivity, as thepopulations at risk for meningococcal disease are characterized bynon-existing or low baseline bactericidal activity to most strains. Inaddition, each of the 4 primary MnB test strains expresses an LP2086variant that is different from the LP2086 variant in the vaccine, thusallowing an objective assessment of functional immunogenicity andefficacy to invasive meningococcal disease (IMD) strains circulating inthe population.

The hSBA measures the amount of anti-meningococcal serogroup B (MnB)antibody in serum capable of initiating complement-mediated bactericidalactivity. Briefly, test serum is serially-diluted in 2-fold steps andadded to 96-well assay plates. MnB SBA test strains and human serumcomplement are added, initiating the bactericidal reaction. Afterincubation of the assay plates at 37° C. for 30-60 minutes (depending onSBA test strain; called T30), the reaction mixture containing bacteriasurviving this incubation are diluted and transferred to microfilterplates. Following overnight incubation, surviving bacteria expressed ascolony-forming units (CFU) are enumerated using an Immunospot Analyzer.The raw CFU data are recorded electronically and transferred to a dataanalysis application that calculates the hSBA titer. The hSBA titer isthe reciprocal of the highest 2-fold dilution of a test serum thatresults in at least a 50% reduction of MnB bacteria (50% bacterialsurvival) compared to the T30 CFU value (i.e., the number of bacteriasurviving after incubation in assay wells containing all assaycomponents except test serum; 100% bacterial survival). Titers may bereported as step titers, i.e., 1:4, 1:8, 1:16, etc. Serum samples aretested by two individual, replicate determinations in the same assay.The final titer reported for samples in which the replicate measurementsare not identical is the lower of the two replicate measurements whensystem suitability and sample suitability criteria (e.g. replicatetiters must agree within one 2-fold dilution) are met.

hSBA assays were done after serially diluting test sera in Dulbecco'sphosphate-buffered saline. Bacteria (roughly 2000 colony-forming units)and human serum complement (20% by weight final concentration) wereadded to the serially diluted sera in 96-well plates and incubated at37° C. for 30-40 min (depending on hSBA test strain) in a small-radiusorbital shaker at 700 rpm. After incubation, a portion of the reactionmixture was transferred to microfilter plates. After overnightincubation, surviving bacteria were counted with an Immunospot Analyzer(Cellular Technology Limited; Shaker Heights, Ohio, USA) and hSBA titerswere analysed with SAS (version 9.2). The hSBA titer was calculated asthe reciprocal of the interpolated test serum dilution that resulted ina 50% reduction of bacteria compared with a control not subjected totest serum (i.e., surviving bacteria at the end of the hSBA reaction).Per protocol hSBAs were done on the basis of the hSBA titer that was ator above the lower limit of quantitation of the hSBA assays asestablished during qualification of the assays with strains listed inthe Table 1 of Example 1.

Human serum is the complement source for the SBA. However, the hSBAtiters may vary depending on the human complement lot used. Accordingly,human complement is preferably controlled through rigorous screening andqualification to ensure consistent performance in the hSBA. For thehSBA, human serum complement may be pooled from multiple normal healthyhuman adults or used from individual donors (i.e., not pooled).

Example 3—Polysorbate-80

Three parameters have been optimized for drug product formulation: pH,aluminum concentration and polysorbate 80 (PS-80) to protein molarratio. In a dose of the composition having a total volume of 0.5 ml,optimal protein binding to aluminum is achieved at a pH of about 6.0 andabout a 0.5 mg/ml concentration of aluminum as aluminum phosphate(AlPO₄) (which is equivalent to 0.25 mg aluminum per dose). The PS-80 toprotein molar ratio is maintained at 2.8±1.4 in order to stabilize theformulation with respect to in vitro potency. Polysorbate 80 (PS-80) isadded to drug substance to obtain the target PS-80 to protein molarratio of 2.8. Therefore, PS-80 is preferably not added during the drugproduct formulation.

Example 4

Randomized, Placebo-Controlled, Phase 2 Study of the Immunogenicity andSafety of REPEVAX® Administered Concomitantly with Bivalent rLP2086Vaccine in Healthy Adolescents

Background/Aims:

The investigational bivalent rLP2086 vaccine, being developed to preventNeisseria meningitidis serogroup B (MnB) disease in adolescents, wasevaluated with concomitant administration of REPEVAX®, adTaP-inactivated polio vaccine (which may be described in U.S. Pat. No.7,479,283, WO1990/013313, and EP1666057 B1, and UK MarketingAuthorisation PL06745/0121) currently used in this population.

Methods:

Adolescents, randomized 1:1 to REPEVAX+rLP2086 or REPEVAX+saline werevaccinated at 0, 2, and 6 months. The proportion of subjects achievingprespecified antibody levels to 9 REPEVAX antigens 30 days after initialvaccination were determined. Immune responses (hSBA) to 4 MnB teststrains were measured 30 days after vaccinations 2 and 3. Adverse events(AE) and local/systemic reactions were assessed.

REPEVAX (Sanofi Pasteur MSD limited) is a combined low-dose diphtheria,tetanus, acellular pertussis, and inactivated poliomyelitis virusvaccine containing diphtheria toxoid (not less than 2 IU), tetanustoxoid (not less than 20 IU), pertussis antigens (pertussis toxoid (2.5micrograms), filamentous haemagglutinin (5 micrograms), pertacti (3micrograms), and fimbriae Types 2 and 3 (5 micrograms)), polio virus(inactivated) type 1 (40 D antigen units), poliovirus (inactivated type2 (8 D antigen units), poliovirus (inactivated) type 3 (32 D antigenunits), adsorbed on aluminum phosphate (1.5 mg (0.33 mg aluminum)) per0.5-mL dose.

Immune responses to the diphtheria, tetanus, and pertussis components ofREPEVAX (diphtheria toxoid, tetanus toxoid, pertussis toxoid, pertactin,fimbriae types 2 and 3 and filamentous haemagglutinin) were assessedusing a multiplexed LUMINEX assay. Immune responses to poliovirus types1, 2, and 3 were measured in virus neutralization assays. Sera obtainedfrom all subjects in both groups were used in these assays.

For assessment of the immune response to bivalent rLP2086, functionalantibodies were analyzed in hSBAs with the 4 primary MnB test strainsdescribed. Four primary MnB hSBA test strains (A22, A56, B44, and B24),2 expressing LP2086 subfamily A and the other 2 expressing LP2086subfamily B variants were selected. These 4 primary hSBA test strains(from 4 of the 6 fHBP phylogenetic subgroups and representing >90% ofdisease isolates circulating in the USA and Europe) were used fordetermination of the primary immunogenicity endpoints in this study.Additionally, the A22, B24, and B44 variants are epidemiologicallyrelevant variants in Europe, while in the US, A22 and B24 are the mostprevalent variants found expressed on disease causing MnB strains. TheMnB hSBAs were validated prior to testing of samples used for theprimary and secondary analyses.

Serum samples from 50% of randomly selected subjects in both groups hadhSBA performed with A22 and B24 and the other 50% were tested with A56and B44. These tests were performed on blood samples collected beforeVaccination 1, after Vaccination 2, and after Vaccination 3.

The immunogenicity of REPEVAX is assessed by using prespecified criteriafor each antigen defined in the pivotal Phase 3 clinical trials inadolescents that formed the basis of licensure for REPEVAX. The REPEVAXconcomitant antigens include diphtheria, tetanus, pertussis toxoid,pertussis filamentous hemagglutinin, pertussis pertactin, pertussisfimbrial agglutinogens type 2+3, poliovirus type 1, poliovirus type 2,poliovirus type 3. The exception is for pertussis fimbrial agglutinogens(FIM) types 2+3, which defined a titer of EU/mL in the assay used forlicensure of REPEVAX. In this study the lower limit of quantification(LLOQ) of the pertussis FIM types 2+3 assay was ≥10.6 EU/mL, which ishigher and therefore more stringent than the licensing criteria ofREPEVAX.

The LLOQs for the concomitant antigens were 0.037 IU/mL for diphtheriatoxoid; 0.05 IU/ml for tetanus toxoid; 0.9 EU/mL for pertussis toxoid;2.9 EU/mL for pertussis filamentous hemagglutinin, 3.0 EU/mL pertussispertactin; 10.6 EU/mL pertussis fimbrial agglutinogens type 2+3; 1:8 forpoliovirus type 1, poliovirus type 2, poliovirus type 3.

Additional descriptive endpoints for the primary objective were theantibodies to concomitant vaccine antigens measured as geometric meantiter (GMTs) or geometric mean concentrations (GMCs) at postvaccination1 (Visit 2).

Another endpoint was the proportion of subjects with hSBA titer ≥LLOQ atPostvaccination 3 (Visit 6) for each of the 4 primary MnB test strains.

Concomitant Vaccine Antigens.

The proportion of subjects achieving the prespecified criteria for theconcomitant vaccine antigens 1 month after vaccination of diphtheria,tetanus, and pertussis acellular (dTaP)-IPV (REPEVAX) was computed witha 2-sided 95% exact (or Clopper-Pearson confidence limit) for Group 1and Group 2. The difference (bivalent rLP2086/dTaP-IPV—dTaP-IPV, orGroup 1-Group 2) of the proportions was also calculated along with a2-sided 95% exact CI for the difference. Noninferiority was declared ifthe lower limit of the 2-sided 95% CI for the difference was greaterthan −0.10 (−10%) for all of the 9 antigens in the dTaP-IPV vaccine.

hSBAs with Primary Test Strains.

For each primary MnB hSBA test strain, the number and proportion ofsubjects achieving hSBA titers ≥LLOQ, ≥1:4, ≥1:8, ≥1:16, and ≥1:128 ateach blood sampling time point were descriptively summarized along withthe exact 2-sided 95% CI (or Clopper-Pearson confidence limit) for theproportion.

Results:

Of 749 subjects randomized, 685 (91.5%) included the evaluableimmunogenicity population. Immune responses following REPEVAX+rLP2086 orREPEVAX+saline were noninferior for all 9 REPEVAX antigens. Immuneresponses to the bivalent rLP2086 vaccine were substantial after 2 dosesand further enhanced after 3 doses (Table 2). Mild-to-moderate injectionsite pain was the most common local reaction; headache and fatigue werethe most common systemic events. The proportion of subjects reporting anAE within 30 days postvaccination was similar (8.8% and 11.4%, forREPEVAX+rLP2086 and REPEVAX+saline, respectively).

For the concomitant vaccine evaluable immunogenicity population, theproportion of subjects achieving the prespecified level of antibodies toconcomitant vaccine antigens (threshold for response) 1 month after theREPEVAX dose was similar between the bivalent rLP2086+REPEVAX group andthe REPEVAX alone group for concomitant vaccine antigens: diphtheriatoxoid (99.4% in each group), tetanus toxoid (100% in each group),pertussis toxoid (94.7% and 96.0%, respectively), pertussis filamentoushemagglutinin (100% in each group), pertussis pertactin (100% in eachgroup), pertussis fimbrial agglutinogens type 2+3 (97.6% and 98.9%,respectively), poliovirus type 1 (100% in each group), poliovirus type 2(100% in each group), poliovirus type 3 (100% in each group).

Noninferiority was achieved because the lower bound of the 2-sided 95%CI for the difference in proportion of responders between the bivalentrLP2086+REPEVAX group (Group 1) and the REPEVAX alone group (Group 2), 1month after the REPEVAX dose was greater than −0.10 (−10%) for the 9antigens in REPEVAX (i.e., the lowest lower bound of the 95% CI on theproportion difference was −4.7% (pertussis toxoid). Hence, the immuneresponse induced by REPEVAX given with bivalent rLP2086 was noninferiorto the immune response induced by REPEVAX alone.

The proportion of subjects with an hSBA titer ≥LLOQ for each of the 4primary MnB test strains for the Postvaccination 3 evaluableimmunogenicity population was assessed. The LLOQ for A22 was an hSBAtiter equal to 1:16 while the LLOQ for all the other MnB test stains wasan hSBA titer equal to 1:8.

For Group 1, the proportion of subjects with an hSBA titer ≥LLOQ atbaseline (before Vaccination 1) was 14.4% for primary MnB strain A22,18.2% for A56, 12.7% for B24, and 6.2% for B44. For Group 2, theproportion of subjects with an hSBA titer ≥LLOQ at baseline (beforeVaccination 1) was 23.0% for primary MnB strain A22, 21.8% for A56,12.9% for B24, and 6.3% for B44.

Substantial hSBA responses were observed among Group 1 subjects afterDose 2 of bivalent rLP2086, with additional increases observed after 3doses 1 month after Vaccination 3. For Group 1 (bivalentrLP2086+REPEVAX), the proportion of subjects achieving an hSBA titer≥LLOQ at 1 month after Vaccination 2 and at 1 month after Vaccination 3was 81.1% and 95.6% for A22, 97.3% and 100% for A56, 81.0% and 96.8% forB24, and 55.5% and 81.5% for B44. While substantial hSBA responses wereachieved after only two bivalent rLP2086 doses, the increase in theproportion of subjects with an hSBA titer ≥LLOQ after 2 doses (1 monthafter Vaccination 2) compared to 3 doses (1 month after Vaccination 3)exemplifies the enhancement of an immune response after 3 doses. In thecontrol group (Group 2), the proportions of subjects with an hSBA titer≥LLOQ for each of the 4 primary MnB test strains at 1 month afterVaccination 2 and 1 month after Vaccination 3 were similar to thebaseline hSBA results for each MnB test strain (before Vaccination 1).

For the 4 primary MnB test strains, the proportion of subjects in Group1 exhibiting a defined hSBA titer was greater after 3 doses than after 2doses. Subjects who achieved an hSBA titer of ≥1:16 are described, sincethis titer is a 4-fold increase from a 1:4 titer (a titer of ≥1:4 iswidely recognized as the correlate of protection against IMD). For Group1, the proportion of subjects with an hSBA titer of 1:16 at 1 monthafter Vaccination 2 was 81.8% for A22, 97.3% for A56, 68.0% for B24, and53.4% for B44. One month after Vaccination 3, the proportion of subjectswith an hSBA titer of 1:16 was 95.6% for A22, 100% for A56, 87.3% forB24, and 79.5% for B44.

In the control group (Group 2), the proportions of subjects exhibitingdefined hSBA titers for each of the 4 primary MnB test strains at 1month after Vaccination 2 and 1 month after Vaccination 3 were similarto the proportion of subjects with the defined hSBA titer at baseline(before Vaccination 1).

For Group 1, the proportion of subjects with an hSBA titer of 1:16following 3 doses of bivalent rLP2086 demonstrated that the vaccineelicits a robust immune response when 3 doses of bivalent rLP2086 wereadministered.

hSBA Geometric Mean Titers (GMTs).

In general, the GMTs at baseline were below the hSBA LLOQs for bothgroups. For Group 1, hSBA GMTs at 1 month after Vaccination 2 were 35.5for A22, 91.1 for A56, 15.9 for B24, and 14.6 for B44. The hSBA GMTs at1 month after Vaccination 3 were 63.4 for A22, 151.5 for A56, 28.3 forB24, and 36.5 for B44.

For Group 1, the observed GMTs after 2 doses for subfamily A strains, aswell as after 3 doses for subfamily B strains, were indicative of arobust immune response.

Reverse cumulative distribution curves (RCDCs) showing the distributionof hSBA titers for A22, A56, B24, and B44 were assessed. Results fromthe RCDCs in Group 1 showed that substantial immune responses wereobserved among Group 1 subjects after Vaccination 2 of bivalent rLP2086,however, the figures also showed the benefit of a third dose of bivalentrLP2086 as greater proportion of subjects achieved higher titers againstthe 4 MnB test strains. The effect was most pronounced for strain B44.

Conclusions: When given concomitantly with bivalent rLP2086, REPEVAXinduced immune responses that were noninferior to those elicited byREPEVAX alone. The bivalent rLP2086 vaccine induced robust bactericidalresponses to four diverse MnB test strains, particularly to thoserepresenting subfamily B, that were greater after 3 doses than 2 doses.Concomitant administration was generally safe and well tolerated.

TABLE 2 Immune response to 4 heterologous MnB test strains after doses 2and 3 of bivalent rLP2086 rLP2086 + REPEVAX Saline + REPEVAX Strain[fHBP variant] hSBA ≥ LLOQ hSBA ≥ LLOQ Time point N^(a) hSBA GMT(95%Cl)^(c) n^(b) (%) (95% Cl)^(d) N^(a) hSBA GMT (95% Cl)^(c) n^(b) (%)(95% Cl)^(d) PMB80 [A22] Dose 2 154  35.5 (30.27, 41.61) 126 (81.8)(74.8, 87.6) 166 11.2 (10.02, 12.46) 36 (21.7) (15.7, 28.7) Dose 3 158 63.4 (55.29, 72.79) 151 (95.6) (91.1, 98.2) 166 11.0 (9.92, 12.27) 33(19.9) (14.1, 26.8) PMB2001 [A56] Dose 2 149  91.1 (78.00, 106.51) 145(97.3) (93.3, 99.3) 151  8.3 (6.76, 10.29) 39 (25.8) (19.1, 33.6) Dose 3148 151.5 (131.47, 174.59) 148 (100.0) (97.5, 100.0) 152  8.5 (6.90,10.54) 40 (26.3) (19.5, 34.1) PMB2948 [B24] Dose 2 153  15.9 (13.55,18.55) 124 (81.0) (73.9, 86.9) 167  4.8 (4.41, 5.19) 20 (12.0) (7.5,17.9) Dose 3 157  28.3 (24.49, 32.66) 152 (96.8) (92.7, 99.0) 170  4.8(4.41, 5.15) 22 (12.9) (8.3, 18.9) PMB2707 [B44] Dose 2 146  14.6 (11.6,18.43)  81(55.5) (47.0, 63.7) 159  4.7 (4.24, 5.12) 12 (7.5) (4.0, 12.8)Dose 3 146  36.5 (28.93, 46.18) 119 (81.5) (74.2, 87.4) 159  4.7 (4.29,5.24) 13 (8.2) (4.4, 13.6) GMT = hgeometric mean titer; hSBA = serumbactericidal assay using human complement; LLOQ = lower limit ofquantitation (titer 1:16 for PMB80 [A22] and 1:8 for the other MnB teststrains); rLP2086 = recombinant lipoprotein 2086. ^(a)Number of subjectswith valid hSBA titers for the given strain ^(b)Number of subjects withhSBA titer ≥ LLOQ for given strain at specified time point^(c)Confidence intervals are back transformations of confidenceintervals based on Student t distribution for the mean logarithm of thehSBA titers ^(d)Exact 2-sided confidence intervals based on observedproportion of subjects using the Clopper and Pearson method

Example 5

Immunogenicity of an Investigational Meningococcal Serogroup B BivalentrLP2086 Vaccine in Healthy Adolescents

Background and Aims:

Neisseria meningitidis serogroup B (MnB) causes invasive disease ininfants, adolescents, and adults. A conserved, surface-exposedlipoprotein, LP2086 (a factor H binding protein [fHBP]), is a promisingMnB vaccine target. Safety and immunogenicity of an investigationalbivalent, recombinant vaccine (rLP2086) were studied in healthyadolescents (11-18 years).

Methods:

Subjects in this placebo-controlled, single-blind study were randomizedto two 3-dose schedules and three 2-dose schedules. Each 120-μg dosecontained 2 rLP2086 antigens, 1 from each LP2086 subfamily (A and B).Saline was given when vaccine was not scheduled. Serum bactericidalassays using human complement (hSBA) were performed with 4 MnB teststrains (heterologous to vaccine fHBP).

Results:

1713 subjects (mean age, 14.4 y) were randomized. One month after 3doses of vaccine, hSBA titers ≥8 to subfamily A and B strains wereobserved in 95-99% and 86-89% of subjects, respectively; after 2 doses,these numbers ranged from 91-100% and 69-77% of subjects, respectively.Of the 2-dose schedules, 0 and 6 months induced the highest antibodyresponses (Table 1 of Example 5). hSBA GMTs after 2 doses ranged from6.2-125.6 and after 3 doses ranged from 25.6-155.6 across the 4 MnBheterologous test strains. Mild-to-moderate injection site pain was themost common local reaction. Fever ≥38° C. was experienced in 3.3-6.5%and 2.1% of rLP2086 and saline recipients, respectively, after dose 1.

TABLE 1 Proportion of Subjects Achieving hSBA T

 for Each Strain 1 Month After Last Dose of Bivalent rLP20 

Group 1 Group 2 Group 3 Group 4 Group 5 (0, 1,

 mo) (0, 2,

 mo) ( 

 mo) (0, 2 mo) (2, 6 mo) Strain n = 

n = 

n = 356-370 n = 234-240 n = 110-113 [

BP %  

% 

% 

% 

% 

variant]

indicates data missing or illegible when filed

TABLE 2 hSBA GMTs for Each Strain 1 Month After Last Dose of

  rLP20 

Group 1 Group 2 Group 3 Group 4 Group 5 (0, 1,

  (0, 2,

  (0,

  (0, 2 (

  mo) mo) mo) mo) mo) Strain n = 354-3

2 n = 3

n =

n =

n =

[

BP GMT 

GMT 

GMT 

GMT 

GMT 

variant]

indicates data missing or illegible when filedConclusions: rLP2086 was well tolerated. All dosing regimens yieldedrobust bactericidal responses that were most pronounced after 3 doses.

Table 1 of Example 5 is the same as Table 1 of Example 1, describedabove. Table 2 summarizes the hSBA GMTs and the corresponding CIs bystudy time for the evaluable immunogenicity population. GMTs increasedfrom baseline (before Injection 1) and continued to increase with eachsubsequent dose of bivalent rLP2086.

For the 4 primary MnB strains, the GMTs were greater after 3 doses ofbivalent rLP2086 (Groups 1 and 2) than after 2 doses (Groups 3, 4, and5). The GMTs were similar between the two 3-dose groups, and they weresimilar among the three 2-dose groups.

Before injection 1 (baseline), hSBA GMTs for Groups 1, 2, 3, 4, and 5were as follows: 7.1, 6.3, 6.4, 6.4, and 6.8 for A22, respectively; 6.8,6.1, 6.7, 6.3, and 6.2 for A56, respectively; 5.3, 5.1, 5.0, 4.9, and5.1 for B24, respectively; and 4.4, 4.5, 4.5, 4.6, and 4.4 for B44,respectively.

For Group 1 (0-, 1-, and 6-month), there was a substantial increase inGMTs noted 1 month after Dose 2 for all 4 primary MnB strains (24.4,77.3, 13.8, and 13.1 for A22, A56, B24, and B44, respectively). The GMTsfurther increased after 3 doses of bivalent rLP2086 for Group 1 subjectsfor the 4 primary MnB test strains; 55.1 (A22); 152.96 (A56); 29.1(B24); and 40.3 (B44).

For Group 2, similar increases in GMTs were noted after 2 and 3 doses ofbivalent rLP2086. GMTs for Group 2 subjects after 2 doses of bivalentrLP2086 were 32.9 for A22; 94.6 for A56; 14.9 for B24; and 15.5 for B44.After 3 doses, the GMTs increased to 56.3 for A22; 155.6 for A56; 25.6for B24; and 35.0 for B44.

For Groups 1 and 2, the observed GMTs after 2 doses for subfamily Astrains, as well as after 3 doses for subfamily B strains, areindicative of a robust immune response.

For Group 3, small increases in GMTs were noted after 1 dose of bivalentrLP2086 as follows: 12.0 for A22; 18.5 for A56; 9.2 for B24; and 5.7 forB44. After 2 doses GMTs increased to 48.4 for A22; 125.6 for A56; 20.6for B24; and 22.5 for B44.

For Group 4, GMTs were 13.3 for A22; 17.7 for A56; 9.8 for B24; and 5.9for B44 after 1 dose of bivalent rLP2086. After 2 doses of bivalentrLP2086, GMTs were 37.1 for A22; 104.9 for A56; 17.7 for B24; and 19.1for B44.

For Group 5, GMTs after 1 dose of bivalent rLP2086 were 16.0 for A22;26.8 for A56; 12.6 for B24; and 6.8 for B44. After 2 doses of bivalentrLP2086, the GMTs increased to 39.6 for A22; 111.8 for A56; 14.7 forB24; and 17.8 for B44.

Taken together, for Groups 3, 4, and 5, the observed GMTs are indicativeof an immune response for subfamily A and B strains after 2 doses ofbivalent rLP2086.

In summary, 3 doses of bivalent rLP2086 provided a robust and thebroadest immune response based on the hSBA titers for the 4 primary MnBtest strains. In comparison to 2 doses, a higher proportion of subjectsreceiving 3 doses of bivalent rLP2086 achieved an hSBA titer ≥1:8 to the4 primary MnB test strains.

The results following the 0-, 1-, and 6-month dosing schedule (Group 1)were similar to the results following the 0-, 2-, and 6-month dosingschedule (Group 2). For Groups 1 and 2, the post-Dose 3 GMT valuesachieved were higher than the post-Dose 2 GMT values. For Groups 1 and2, the post-Dose 2 GMT values ranged from 24.4 to 94.6 for subfamily Astrains and from 13.1 to 15.5 for subfamily B strains. The post-Dose 3GMT values ranged from 55.1 to 155.6 for subfamily A strains and from25.6 to 40.3 for subfamily B strains. For Groups 1 and 2, a higherproportion of subjects achieved an hSBA titer ≥1:8 to the 4 primary MnBtest strains following 3 doses of bivalent rLP2086 when compared to theproportion of subjects achieving an hSBA titer ≥1:8 to the 4 primary MnBtest strains after 2 doses of bivalent rLP2086.

Subjects who achieved an hSBA titer of ≥1:16 were also assessed. ForGroup 1, the percentage of subjects who achieved an hSBA titer of ≥1:16one month after 2 doses of bivalent rLP2086 was 73.5% for A22; 96.3 forA56; 57.6 for B24; and 47.2% for B44. Following 3 doses of bivalentrLP2086, the percentage of subjects in Group 1 who achieved an hSBAtiter of ≥1:16 was 91.4% for A22; 99.2% for A56; 82.8% for B24; and84.8% for B44.

For Group 2, the percentage of subjects who achieved an hSBA titer of≥1:16 one month after 2 doses of bivalent rLP2086 was 88.1% for A22;97.9% for A56; 63.5% for B24; and 58.6% for B44. Following 3 doses ofbivalent rLP2086, the percentage of subjects in Group 2 who achieved anhSBA titer of ≥1:16 was 95.0% for A22; 98.9% for A56; 83.6% for B24; and83.8% for B44.

For Groups 1 and 2, the percentage of subjects achieving an hSBA titerof ≥1:16 following 3 doses of bivalent rLP2086 demonstrated that thevaccine elicits a robust immune response.

For Group 3, the percentage of subjects who achieved an hSBA titer of≥1:16 after 2 doses of bivalent rLP2086 was 93.2% for A22; 98.4% forA56; 73.8% for B24; and 70.8% for B44.

For Group 4, the percentage of subjects who achieved an hSBA titer of≥1:16 one month after 2 doses of bivalent rLP2086 was 90.8% for A22;99.2% for A56; 67.1% for B24; and 64.5% for B44.

For Group 5, the percentage of subjects who achieved an hSBA titer of≥1:16 after 2 doses of bivalent rLP2086 was 91.0% for A22; 99.1% forA56; 64.5% for B24; and 66.7% for B44.

For Groups 3, 4, and 5, the percentage of subjects achieving an hSBAtiter of ≥1:16 demonstrated that the vaccine elicits a robust immuneresponse to subfamily A strains following only 2 doses. However, 3 dosesincreases the robustness of response to subfamily B strains.

The percentage of subjects achieving an hSBA titer of ≥1:16 after 3doses of bivalent rLP2086 shows that the vaccine elicits a robust andbroad immune response to MnB strains expressing LP2086 variants that aredifferent from the vaccine components.

Reverse cumulative distribution curves (RCDCs) showing the distributionof hSBA titers by study times were also assessed for the evaluableimmunogenicity populations for each strain by group. The RCDCs showrobust immune responses after 2 doses of bivalent rLP2086 subfamily Astrains. Following the third dose of bivalent rLP2086, the area underthe response curves increases for all 4 primary MnB test strains,thereby demonstrating the enhancement of the immune response after 3doses of bivalent rLP2086.

The results from the primary and secondary immunogenicity endpointanalyses show that the vaccine can generate antibodies with significanthSBA activity against heterologous subfamily A and subfamily B variantsof MnB. While the proportion of subjects achieving an hSBA titer ≥1:8was higher after 2 or 3 doses of bivalent rLP2086, a large proportion ofsubjects achieved an hSBA titer ≥1:8 one month after 1 dose of bivalentrLP2086. See Group 5 for example.

For the 4 primary MnB test strains, the GMTs were greater after 3 dosesof bivalent rLP2086 (Groups 1 and 2) than after 2 doses (Groups 3, 4,and 5). The GMTs were similar in the two 3-dose groups. The GMTs werealso similar among the three 2-dose groups. These data also demonstraterobust hSBA responses after 3 doses of bivalent rLP2086 based on thepercentages of subjects achieving an hSBA titer ≥1:16.

These data demonstrate that the final formulation of bivalent rLP2086generates a robust immune response and is safe and well tolerated whengiven in 2 or 3 doses. Even 1 dose of bivalent rLP2086 provides asubstantial immune response above baseline and is also safe and welltolerated. Overall, there was no clinically meaningful difference in thesafety profile after 2 or 3 doses of bivalent rLP2086.

Example 6

Safety, Tolerability, and Immunogenicity of a Meningococcal Serogroup BBivalent rLP2086 Vaccine in Healthy Adolescents Aged 11 to 18 Years inThree Phase 2, Randomized, Controlled Studies

Background:

Neisseria meningitidis serogroup B (MnB) is a major cause of invasivemeningococcal disease in adolescents. A conserved, surface-exposedlipoprotein, LP2086 (factor H binding protein [fHBP]), is a promisingvaccine target to protect against invasive disease caused by MnB.Safety, tolerability, and immunogenicity of an investigational bivalent,recombinant MnB vaccine (including SEQ ID NO: 1 and SEQ ID NO: 2, 2.8molar ratio polysorbate-80, 0.5 mg/ml aluminum, 10 mM histidine, and 150mM sodium chloride, herein referred to throughout the Examples as“bivalent rLP2086”) were examined in three phase 2, randomized,controlled studies in healthy adolescents 11-18 years of age.

Methods:

Study 1012 examined 5 vaccine regimens of bivalent rLP2086, whereasstudies 1010 and 1011 evaluated a 3-dose schedule of bivalent rLP2086vaccine given concomitantly with the TdaP-IPV and HPV-vaccines,respectively. Each dose of bivalent rLP2086 contained 60 μg of therLP2086 subfamily A variant A05 and 60 μg of the rLP2086 subfamily Bvariant B01. To examine immunogenicity of bivalent rLP2086 in each ofthe three studies, serum bactericidal assays using human complement(hSBA) were performed with 4 MnB test strains expressing theheterologous fHBP variants A22, A56, B24 and B44, which were selected torepresent relevant diversity of fHBP variability, as well as to providea perspective on the breadth of the vaccine-elicited immune responseagainst strain expressing epidemiologically prevalent fHBP variants.Adverse events and solicited local and systemic reactions were assessed.

Results:

82-100% of subjects in all 3 studies achieved hSBA titers above thelower limit of quantification (LLOQ) for each of the 4 MnB test strains1 month after dose 3 (Table). Across the three studies, the majority ofsystemic events and local reactions were mild to moderate in severity;adverse events were generally not serious or related to the studyvaccine.

Conclusions: Serum bactericidal antibody titers above 1:4 protectagainst invasive meningococcal disease. The demonstration of hSBA titers≥LLOQ to 4 MnB test strains, each heterologous to vaccine antigen, ineach of these adolescent phase 2 studies, suggest that the bivalentrLP2086 vaccine provided a functional antibody response that may bebroadly active against diverse MnB disease-associated strains.Vaccinations with the bivalent rLP2086 were generally well tolerated.

TABLE Proportion of Subjects Achieving an hSBA Titer ≥ LOQ for Each fHBPVariant Expressed by Each Test Strain 1 Month After the Last Dose of theBivalent rLP2086 Vaccine fH8P variant expressed % of Subjects by hSBAtest strain A22 A56 824 844 Study 1012 (dosing regimen) Group 1 (0, 1, 6mo); n = 354-360 91.4 99.4 89.0 88.5 Group 2 (0, 2, 6 mo); n = 352-35995.0 98.9 88.4 86.1 Group 3 (0, 6 mo); n = 356-370 93.2 98.4 81.1 77.5Group 4 (0, 2 mo); n = 234-240 90.8 100.0 73.0 70.1 Group 5 (0, 4 mo); n= 110-113 91.0 99.1 69.1 73.0 Study 1010 (dosing regimen: 0, 2, 6 mo)rLP2086 + TdaP-IPV Vaccine; n = 146-158 95.6 100.0 96.8 81.5 Study 1011(dosing regimen: 0, 2, 6 mo) rLP20864 + HPV Vaccine; n = 833-849 94.098.9 90.5 82.7 rLP2086 + Saline; n = 847-848 96.3 99.4 92.6 85.7 LLOQ =lower limit of quantification; fHBP = factor H binding protein; hSBA =serum bactericidal assays using human complement; TdaP-IPV Vaccine =Tetanus, Diphtheria: Pertussis, Polio Vaccine. LLOQ = the lowest amountof an analyte in a sample that can be quantitatively determined. hSBAtiter s ≥ 0 1:4 area correlate of protection for invasive meningococcaldisease. hSBA titers ≥ LLOQ are above the minimal correlate. LLOQ was1:16 for A22; and 1:8 for A56, B24, and 844.

Example 7

Immunogenicity of a Meningococcal Serogroup B Bivalent rLP2086 Vaccinein Healthy Adolescents Aged 11 to 18 when Administered Concomitantlywith Human Papillomavirus Vaccine

This Phase 2, randomized, observer-blind, controlled study evaluated theimmunogenicity of bivalent rLP2086 with or without coadministration withGARDASIL®, which is a quadrivalent vaccine against human papillomavirus(HPV4) (as also described in U.S. Pat. No. 5,820,870), in healthyadolescents ω11 to <18 years of age. GARDASIL contains recombinantantigens of HPV type 6, 11, 16, and 18 (i.e., HPV-6, HPV-11, HPV-16, andHPV-18) L1 protein. An endpoint was the hSBA GMTs for each of the 4primary MnB test strains at each applicable blood sampling time point.

Methods:

Subjects received bivalent rLP2086 (including SEQ ID NO: 1 and SEQ IDNO: 2, 2.8 molar ratio polysorbate-80, 0.5 mg/ml aluminum, 10 mMhistidine, and 150 mM sodium chloride)+HPV4 (Group 1), bivalentrLP2086+saline (Group 2), or HPV4+saline (Group 3) at months 0, 2, and6. Sera from subjects in Groups 1 and 2 before vaccination 1, and 1month after vaccinations 2 and 3, were tested by serum bactericidalassay using human complement (hSBA) using 4 MnB test strains, eachexpressing an fHBP (A22, A56, B44, and B24) that is heterologous to thevaccine components and represents the breadth of fHBP diversity, as wellas epidemiological prevalence. Endpoints assessed included theproportion of subjects with hSBA titers the lower limit of quantitation(LLOQ; 1:16 [A22] or 1:8 [A56, B44, B24]) and hSBA geometric mean titers(GMTs).

To demonstrate noninferiority of administrating GARDASIL plus bivalentrLP2086 compared to GARDASIL alone, immunogenicity assessments wereperformed with 2 hSBAs, using 1 primary test strain representingsubfamily A variants (A22) and 1 primary test strain representingsubfamily B variants (B24). However, all 4 primary MnB test strains wereused for determination of additional bivalent rLP2086immunogenicity/efficacy exploratory endpoints.

For assessment of the immune response to bivalent rLP2086, functionalantibodies were analyzed in hSBAs with meningococcal serogroup B strainsrandomly selected from Pfizer's representative MnB SBA strain pool, asdescribed in Example 2. The hSBAs measured the functional antibodies inhuman sera that in a complement-dependent manner kill the targetmeningococcal strain.

Results:

814 and 812 subjects included the evaluable immunogenicity populationfor Groups 1 and 2, respectively. Compared with before vaccination 1,the proportion of subjects with hSBA titers ≥LLOQ against all 4 teststrains was higher after vaccinations 2 (55%-99%) and 3 (83%-99%; FIG.1). Table A of Example 7 presents the hSBA GMTs for each of the 4primary MnB strains and the corresponding CIs by sampling time point forthe evaluable immunogenicity population. The GMTs at baseline were belowthe hSBA LLOQs for both groups. GMTs ranged from 11.1-70.6 and 11.9-76.3after vaccination 1, and 25.8-117.2 and 28.0-128.2 after vaccination 2in Groups 1 and 2, respectively (Table A below).

For the evaluable immunogenicity population, the hSBA GMTs to the 2primary MnB strains at 1 month after the Vaccination 3 bivalent rLP2086dose for Group 1 and Group 2 were as follows: 53.3 and 57.8,respectively for A22 and 25.8 and 28.0, respectively for B24.

For Group 2 (bivalent rLP2086+saline), hSBA GMTs at 1 month afterVaccination 2 were 33.7 for A22, 76.3 for A56, 16.3 for B24, and 11.9for B44. The hSBA GMTs at 1 month after Vaccination 3 were 57.8 for A22,128.2 for A56, 28.0 for B24, and 31.9 for B44.

For Group 1 (bivalent rLP2086+GARDASIL), hSBA GMTs at 1 month afterVaccination 2 were 31.9 for A22, 70.6 for A56, 15.0 for B24, and 11.1for B44. The hSBA GMTs at 1 month after Vaccination 3 were 53.3 for A22,117.2 for A56, 25.8 for B24, and 27.2 for B44.

Reverse cumulative distribution curves (RCDCs) showing the distributionof hSBA titers for A22, A56, B24, and B44 were assessed for Group 1 andGroup 2 at all sampling time points for the evaluable immunogenicitypopulation. The RCDCs showed that the majority of subjects respondedafter Vaccination 2 and had an additional increase in titer for the 4primary MnB test strains after Vaccination 3. Immune responses to theantigens were similar for Groups 1 and 2.

Conclusions: Bivalent rLP2086 can be administered with HPV4 withoutaffecting the bactericidal response assessed by hSBA seroresponse orGMTs. Since hSBA titers≥1:4 correlate with protection againstmeningococcal disease, these data indicate the potential for protectionof adolescents against a broad range of MnB strains followingadministration of the bivalent rLP2086 in the setting of concomitantadministration of HPV vaccine.

TABLE A hSBA GMTs - Evaluable Immunogenicity Population Strain [Variant]Group 1 Group 2 Sampling Time rLP2086 + HPV4 rLP2086 + Saline Pointn^(a) GMT^(b) (95% Cl)^(c) n^(a) GMT^(b) (95% Cl)^(c) PMB80 [A22] Before794 9.6 (9.3, 10.0) 799 9.9 (9.5, 10.3) Vaccination 1 1 Month After 79431.9 (29.96, 33.94) 801 33.7 (31.69, 35.85) Vaccination 2 1 Month After803 53.3 (50.22, 56.66) 801 57.8 (54.44, 61.44) Vaccination 3 PMB2001[A56] Before 757 5.0 (4.78, 5.32) 740 5.0 (4.75, 5.28) Vaccination 1 1Month After 790 70.6 (66.17, 75.34) 795 76.3 (71.93, 80.99) Vaccination2 1 Month After 796 117.2 (110.14, 124.76) 802 128.2 (120.65 136.27)Vaccination 3 PMB2948 [B24] Before 801 4.3 (4.23, 4.46) 793 4.5 (4.35,4.65) Vaccination 1 1 Month After 770 15.0 (13.88, 16.15) 770 16.3(15.15, 17.62) Vaccination 2 1 Month After 788 25.8 (24.14, 27.56) 79328.0 (26.24, 29.87) Vaccination 3 PMB2702 [B44] Before 806 4.1 (4.04,4.15) 805 4.2 (4.10, 4.31) Vaccination 1 1 Month After 783 11.1 (10.21,12.01) 776 11.9 (10.94, 12.96) Vaccination 2 1 Month After 799 27.2(24.99, 29.68) 795 31.9 (29.25, 34.82) Vaccination 3 GMT = geometricmean titer; HPV4 = quadrivalent human papillomavirus vaccine; hSBA =serum bactericidal assay using human complement. ^(a)n = number ofsubjects with valid and determinate hSBA titers for the given strain.^(b)Geometric mean titers were calculated using all subjects with validand determinate hSBA titers at the given time point. ^(c)Confidenceintervals are back transformations of confidence intervals based on theStudent t distribution for the man logarithm of the hSBA titers.

Example 8

Immunogenicity of Human Papilloma Vaccine Coadministered with a BivalentrLP2086 Vaccine Against Meningococcal Serogroup B in Healthy Adolescents

Background:

This Phase 2, randomized study evaluated coadministration of aquadrivalent vaccine against human papillomavirus (HPV4), with bivalentrLP2086, an investigational vaccine against invasive disease caused byNeisseria meningitidis serogroup B (MnB), in healthy adolescents ≥11 to<18 years of age.

Methods:

Subjects received HPV4+bivalent rLP2086 (Group 1), bivalentrLP2086+saline (Group 2), or saline+HPV4 (Group 3) at months 0, 2, and6. Sera were collected at baseline and after doses 2 and 3 in allgroups. Immune responses to HPV4 antigens (HPV-6, 11, 16, and 18) weredetermined by competitive LUMINEX immunoassays (cLIAs). Bivalent rLP2086immunogenicity was measured by serum bactericidal assay using humancomplement (hSBA) with 2 MnB test strains expressingvaccine-heterologous fHBP variants (A22 and B24). Immunogenicityendpoints, all after dose 3, included: geometric mean titers (GMTs)against HPV antigens in Groups 1 and 3; hSBA GMTs for strains expressingvariants A22 and B24 in Groups 1 and 2; and seroconversion rate for HPVantigens in baseline seronegative subjects in Groups 1 and 3. Safety ofbivalent rLP2086 was also assessed after concomitant administration withHPV4 or saline.

Assessments of the immune response to GARDASIL (HPV type 6, 11, 16, and18 L1 protein) were performed using cLIAs based on a fluorescentlylabeled microsphere-based platform (LUMINEX). Sera obtained from allsubjects in Groups 1 and 3 prior to the first vaccination with GARDASIL(Visit 1) and 1 month after the third vaccination with GARDASIL (Visit5) were used in these assays.

The comparison of the GMTs to the 4 HPV antigens for Group 1 and Group3, with their corresponding GMT ratios (GMRs) of Group 1 to Group 3 andthe 2-sided 95% CIs of the ratios is presented in Table A below of thepresent Example. The criterion for the noninferiority margin was1.5-fold, which corresponds to a value of 0.67 for the lower limit ofthe 2-sided 95% CI of the GMR. The 1.5-fold criterion of 0.67 was metfor all the MnB test strains and the HPV antigens except for HPV-18,which had a lower bound 95% confidence interval (CI) of 0.62. In aseparate analysis, ≥99% of subjects seroconverted to all 4 HPV antigensin both the Saline+HPV4 and rLP2086+HPV4 groups.

Another objective of this study was to describe the immune responseinduced by bivalent rLP2086+GARDASIL (Group 1) and by saline+GARDASIL(Group 3), as measured by seroconversion in the HPV immunogenicityassays after the Vaccination 3 dose of GARDASIL (Visit 5) in bothgroups.

The seroconversion rate for each of the 4 HPV antigens, 1 month afterthe last dose of GARDASIL for subjects who were HPV-seronegative atbaseline in Group 1 and Group 3, was calculated as the proportion ofsubjects with anti-HPV serum cLIA levels ≥20 mMU/ml for HPV-6, ≥16mMU/ml for HPV-11, ≥20 mMU/ml for HPV-16, and ≥24 mMU/ml for HPV-18.

The number and proportion of baseline HPV-seronegative subjectsachieving the prespecified criteria for seroconversion for the 4 HPVantigens with the corresponding 95% CIs in each group, the percentdifferences (Group 1-Group 3) in the proportion, and the 95% CIs of thedifferences are presented in Table B of Example 8 for the baselineHPV-seronegative evaluable immunogenicity population.

Results:

The prespecified noninferiority criteria set at 1.5-fold (0.67 lowerlimit of 95% CI for GMRs) were met for 3 of 4 HPV antigens (not HPV-18)and both MnB test strains (Table A). Seroconversion rates in Groups 1and 3 were ≥99% for all HPV antigens (Table B). Greater localreactogenicity occurred after rLP2086 compared with saline but did notincrease with later doses; injection site pain was the most common localreaction. Systemic events in all 3 groups were generally mild andmoderate in severity.

For the evaluable immunogenicity population, the GMTs of antibodies tothe 4 HPV antigens at 1 month after the GARDASIL dose at Vaccination 3for Group 1 and Group 3 were as follows: 451.8 and 550.3, respectively(HPV-6); 892.9 and 1084.3, respectively (HPV-11); 3695.4 and 4763.4,respectively (HPV-16); and 744.0 and 1047.4, respectively (HPV-18). TheGMRs of Group 1 to Group 3 at 1 month after the GARDASIL dose atVaccination 3 were 0.82 for HPV-6 (95% CI: 0.72, 0.94), 0.82 for HPV-11(95% CI: 0.74, 0.91), 0.78 for HPV-16 (95% CI: 0.68, 0.88), and 0.71 forHPV-18 (95% CI: 0.62, 0.81). Therefore, the lower limits of the 2-sided95% CIs for anti-HPV GMRs for Group 1 compared with Group 3 were 0.72for HPV-6, 0.74 for HPV-11, 0.68 for HPV-16, and 0.62 for HPV-18. The1.5-fold criterion of 0.67 (the lower limit of the 2-sided 95% CI of theGMR) was met for all HPV antigens except for HPV-18, which had a lowerbound of the 95% CI of 0.62.

The GMRs of the bivalent rLP2086+GARDASIL group to the bivalentrLP2086+saline group at 1 month after the Vaccination 3 bivalent rLP2086dose were 0.92 for A22 (95% CI: 0.85, 1.00), and 0.92 for B24 (95% CI:0.84, 1.01). The lower limits of the 2-sided 95% CIs for the hSBA GMRsfor Group 1 compared with Group 2 were 0.85 for A22 and 0.84 for B24,which are both greater than 0.67 and therefore met the noninferioritymargin of 1.5-fold.

The data from bivalent rLP2086+GARDASIL (Group 1) administration werecompared to data from the bivalent rLP2086+saline (Group 2)administration by analyzing the hSBA titer 4-fold response rates for 2primary MnB strains (A22 and B24) at 1 month after Vaccination 3 Theproportions of subjects achieving ≥4-fold rise in hSBA titer frombaseline to 1 month after Vaccination 3 for the 2 primary MnB strainswere measured for both Group 1 subjects who received bivalentrLP2086+GARDASIL and Group 2 subjects who received bivalentrLP2086+saline. Of the subjects in Group 1, 85.3% exhibited ≥4-fold risein hSBA titers against B24. Of the subjects in Group 2, 86.4% exhibited≥4-fold rise in hSBA titers against A22, and 84.8% exhibited ≥4-foldrise in hSBA titers against B24.

The difference in the proportion of responders between Group 1 and Group2 at 1 month after Vaccination 3 was −1.1% for A22 (95% CI: −4.6, 2.3)and −1.4% for B24 (95% CI: −5.1, 2.3). The differences of 4-foldresponse rates were all near a value of 1%, with the lower bounds of the95% CI of the proportion difference being −4.6% A22 and −5.1% B24.

The noninferiority criteria of bivalent rLP2086+GARDASIL compared tosaline+GARDASIL or compared to bivalent rLP2086+saline required that thelower limit of the 2-sided 95% CIs for the GMRs for antibodies to HPVfor all 4 HPV antigens (HPV-6, HPV-11, HPV-16, and HPV-18) and for hSBAtiters using 2 primary MnB test strains (A22 and B24) 1 month afterVaccination 3 be greater than 0.67. This prespecified criterion was metfor both MnB test strains and at least 3 of the 4 HPV antigens. ForHPV-18, the lower limit of the 2-sided CIs for the GMR was slightlybelow the prespecified threshold of 0.67, at 0.62.

The 4-fold rise responses to 2 primary MnB test strains (A22 and B24)were similar (ranged from 83.4% to 86.4%) between the group thatreceived bivalent rLP2086+GARDASIL and the group that received bivalentrLP2086+saline.

The proportions of subjects in Groups 1 and 2 with prevaccination (i.e.,before Vaccination 1) hSBA titers of ≥1:4 were 15.2% and 18.8%,respectively, for strain A22; 10.4% and 10.5%, respectively, for strainA56; 6.1% and 8.4%, respectively, for strain B24; and 1.7% and 3.2%,respectively for strain B44. In addition, the proportions of subjects inGroups 2 and 1 with prevaccination hSBA titers of ≥1:16 were 13.7% and16.4%, respectively for strain A22; 9.0% and 9.1%, respectively, forstrain A56; 4.1% and 5.4%, respectively, for strain B24; and 1.2% and2.1%, respectively, for strain B44.

In Group 2 (bivalent rLP2086+saline), the proportion of subjects with anhSBA titer ≥1:4 at 1 month after Vaccination 2 was 86.3% for A22, 98.7%for A56, 77.1% for B24, and 60.1% for B44. One month after Vaccination3, the proportion of subjects with an hSBA titer of ≥1:4 was 96.4% forA22, 99.4% for A56, 92.8% for B24, and 86.5% for B44. In Group 1(bivalent rLP2086+GARDASIL), the proportion of subjects with an hSBAtiter of ≥1:4 at 1 month after Vaccination 2 was 83.8% for A22, 97.8%for A56, 71.9% for B24, and 57.7% for B44. One month after Vaccination3, the proportion of subjects with an hSBA titer of ≥1:4 was 94.3% forA22, 99.1% for A56, 91.1% for B24, and 84.4% for B44.

In Group 2 (bivalent rLP2086+saline), the proportion of subjects with anhSBA titer ≥1:16 at 1 month after Vaccination 2 was 85.8% for A22, 98.4%for A56, 68.8% for B24, and 49.9% for B44. One month after Vaccination3, the proportion of subjects with an hSBA titer of ≥1:16 was 96.3% forA22, 99.4% for A56, 89.2% for B24, and 82.4% for B44. In Group 1(bivalent rLP2086+GARDASIL), the proportion of subjects with an hSBAtiter of ≥1:16 at 1 month after Vaccination 2 was 83.0% for A22, 97.2%for A56, 65.2% for B24, and 46.4% for B44. One month after Vaccination3, the proportion of subjects with an hSBA titer of ≥1:16 was 94.0% forA22, 98.9% for A56, 86.3% for B24, and 78.0% for B44.

For both Group 1 and Group 2, a high proportion of subjects achieved anhSBA titer of ≥1:16 or greater following 2 or 3 doses of bivalentrLP2086, while most of the subjects had no measureable hSBA titer to anyof the primary MnB test strains at prevaccination Visit 1.

For the baseline HPV-seronegative evaluable immunogenicity population,the proportion of subjects achieving the prespecified criteria for HPVseroconversion for the HPV antigens at 1 month after the GARDASIL doseat Vaccination 3 for the bivalent rLP2086+GARDASIL group (Group 1) andthe saline+GARDASIL group (Group 3) were as follows: HPV-6 (99.4% and99.3%, respectively), HPV-11 (99.6% and 99.5%, respectively), HPV-16(99.6% and 99.5%, respectively), and HPV-18 (99.5% and 99.0%,respectively).

The difference in proportion of responders between the bivalentrLP2086+GARDASIL group (Group 1) and the saline+GARDASIL group (Group 3)at 1 month after the GARDASIL dose was 0.1% for HPV-6 (95% CI; −0.9,1.5), 0.1% for HPV-11 (95% CI: −0.7, 1.3), 0.1% for HPV-16 (95% CI;−0.7, 1.3), and 0.5% for HPV-18 (95% CI; −0.6, 1.9).

For the bivalent rLP2086+GARDASIL group (Group 1) and thesaline+GARDASIL group (Group 3), the seroconversion rate differenceswere within 0.1% and 0.5% across all 4 HPV antigens and theseroconversion rates were very similar across groups, with greater than99% of subjects seroconverting for all 4 HPV antigens.

As an additional evaluation, bivalent rLP2086+GARDASIL (Group 1) wascompared to bivalent rLP2086+saline (Group 2), by analyzing the hSBAtiter 4-fold response rates for 2 primary MnB strains (A22 and B24) at 1month after Vaccination 3. The proportions of subjects achieving an hSBAtiter fold rise ≥4 from baseline to 1 month after Vaccination 3 for the2 primary MnB strains are as follows: Of the subjects in Group 1, 85.3%exhibited ≥4-fold rise in hSBA titers against test strain A22, and 83.4%exhibited ≥4-fold rise in hSBA titers against test strain B24. Of thesubjects in Group 2, 86.4% exhibited ≥4-fold rise in hSBA titers againsttest strain A22 and 84.8% exhibited ≥4-fold rise in hSBA titers againsttest strain B24.

The difference in the proportion of responders between Group 1 and Group2 at 1 month after Vaccination 3 was −1.1% for A22 (95% CI: −4.6, 2.3)and −1.4% for B24 (95% CI: −5.1, 2.3). The differences of 4-foldresponse rate were all near a value of 1%, with the lower bounds of the95% CI of the proportion difference being −4.6% (A22) and −5.1% (B24).

Immune Responses to Bivalent rLP2086.

Another objective of this study was to describe the immune response asmeasured by hSBA performed with 4 primary MnB test strains, 2 expressingLP2086 subfamily A proteins (A22 and A56) and 2 expressing LP2086subfamily B proteins (B24 and B44), measured 1 month after the secondvisit (Visit 3) and the third (Visit 5) vaccinations with bivalentrLP2086.

One of the endpoints for this objective was the proportion of subjectswith hSBA titers≥LLOQ at 1 month after Vaccination 2 (Visit 3) and at 1month after Vaccination 3 (Visit 5) for each of the 4 primary MnB teststrains. The proportion of subjects with hSBA titer ≥LLOQ for each ofthe 4 primary MnB test strains for the evaluable immunogenicitypopulation was assessed. The LLOQ for A22 was an hSBA titer equal to1:16, while the LLOQ for all the other MnB test strains was an hSBAtiter equal to 1:8.

For Group 2 (bivalent rLP2086+saline), the proportion of subjects withan hSBA titer ≥LLOQ at baseline (before Vaccination 1) was 16.4% forA22, 9.3% for A56, 6.9% for B24, and 2.5% for B44. For Group 2, theproportions of subjects achieving an hSBA titer ≥LLOQ at 1 month afterVaccination 2 and at 1 month after Vaccination 3 were 85.8% and 96.3%,respectively, for A22; 98.5% and 99.4%, respectively, for A56; 74.2% and92.6%, respectively for B24; and 57.1% and 85.7%, respectively, for B44.

For Group 1 (bivalent rLP2086+GARDASIL), the proportion of subjects withan hSBA titer ≥LLOQ at baseline (before Vaccination 1) was 13.7% forA22, 9.2% for A56, 5.1% for B24, and 1.4% for B44. For Group 1, theproportions of subjects achieving an hSBA titer ≥LLOQ at 1 month afterVaccination 2 and at 1 month after Vaccination 3 were 83.0% and 94.0%,respectively, for A22; 97.5% and 98.9%, respectively, for A56; 70.6% and90.5%, respectively for B24; and 54.5% and 82.7%, respectively, for B44.

Substantial hSBA responses to the 4 primary MnB test strains wereobserved among both Group 1 and Group 2 subjects at 1 month afterVaccination 2, with additional increases observed at 1 month afterVaccination 3.

The proportion of subjects achieving an hSBA titer fold rise ≥4 for eachof the 4 primary MnB test strains and the proportions of subjectsachieving the composite response for the evaluable immunogenicitypopulation were assessed. The proportions of subjects with an observedhSBA titer ≥LLOQ for all 4 MnB strains combined at baseline (beforeVaccination 1) were similar between Group 1 (0.3%) and Group 2 (0.7%).

For Group 2 (bivalent rLP2086+saline), the proportion of subjectsachieving an hSBA titer fold rise ≥4 from baseline to 1 month afterVaccination 3 was 86.4% for A22, 95.3% for A56, 84.8% for B24, and 80.7%for B44, and 83.9% of subjects achieved a composite hSBA response(hSBA≥LLOQ for all 4 primary strains combined). At 1 month afterVaccination 2, the proportion of subjects achieving an hSBA titer foldrise ≥4 from baseline was 74.2% for A22, 92.6% for A56, 63.4% for B24,and 47.4% for B44, and 51.9% of subjects achieved a composite hSBAresponse.

For Group 1 (bivalent rLP2086+saline), the proportion of subjectsachieving an hSBA titer fold rise ≥4 from baseline to 1 month afterVaccination 3 was 86.4% for A22, 95.3% for A56, 84.8% for B24, and 80.7%for B44, and 83.9% of subjects achieved a composite hSBA response(hSBA≥LLOQ for all 4 primary strains combined). At 1 month afterVaccination 2, the proportion of subjects achieving an hSBA titer foldrise ≥4 from baseline was 74.2% for A22, 92.6% for A56, 63.4% for B24,and 47.4% for B44, and 51.9% of subjects achieved a composite hSBAresponse.

Additional hSBA Fold Response.

Other endpoints were the proportion of subjects achieving at least2-fold and 3-fold hSBA titer increases from baseline to eachpostvaccination blood sampling visit for each of the 4 primary MnBstrains. Note that the LLOQ for A22 was an hSBA titer equal to 1:16,while the LLOQ for all the other MnB test strains was an hSBA titerequal to 1:8.

The proportion of subjects achieving a ≥2-fold rise in hSBA titer frombaseline to 1 month after Vaccination 2 for Group 1 and Group 2 for MnBstrains were 77.3% and 81.1%, respectively, for A22; 94.4% and 95.3%,respectively, for A56; 63.0% and 66.0%, respectively, for B24; and 46.1%and 48.6%, respectively, for B44. The proportions of subjects achievingan hSBA titer fold rise ≥2 from baseline to 1 month after Vaccination 3for Group 1 and Group 2 for MnB strains were 90.2% and 92.8%,respectively, for A22; 97.2% and 97.9%, respectively, for A56; 84.6% and87.2%, respectively, for B24; and 77.7% and 81.7%, respectively, forB44.

The proportions of subjects achieving an hSBA titer fold rise ≥3 frombaseline to 1 month after Vaccination 2 for Group 1 and Group 2 for MnBstrains were 73.1% and 74.2%, respectively, for A22; 92.5% and 92.6%,respectively, for A56; 61.3% and 63.4%, respectively, for B24; and 45.7%and 47.4%, respectively, for B44. The proportions of subjects achievingan hSBA titer fold rise ≥3 from baseline to 1 month after Vaccination 3for Group 1 and Group 2 for MnB strains were 85.3% and 86.4%,respectively, for A22; 95.0% and 95.3%, respectively, for A56; 83.4% and84.8%, respectively, for B24; and 77.0% and 80.7%, respectively, forB44.

In summary of the descriptive endpoints under the objectives, themajority of subjects achieved an hSBA titer ≥LLOQ for both Group 1(bivalent rLP2086+GARDASIL) and group 2 (bivalent rLP2086+saline) forall 4 primary MnB test strains, while only a very small proportion ofsubjects had measurable hSBA titers ≥LLOQ at baseline (prevaccinationVisit 1). Substantial immune responses with the 4 MnB strains wereobserved at 1 month after Vaccination 2, with additional increasesobserved at 1 month after Vaccination 3 for both Group 1 and Group 2subjects. This conclusion was confirmed by the proportion of subjectswith an hSBA titer of ≥1:16 following 3 doses, the observed GMTsachieved after 2 doses and after 3 doses in both groups, and the RCDCsfor the 4 primary MnB test strains.

For both Group 1 and Group 2, a high proportion of subjects achieved anhSBA titer fold ≥4 rise for each of the primary MnB test strains and acomposite hSBA response ≥LLOQ for all 4 primary MnB strains after thethird study vaccination.

In addition, the majority of subjects achieved an hSBA titer fold rise≥3 and an hSBA titer fold rise ≥2 for the 4 primary MnB strains at allsampling time points for both Group 1 (bivalent rLP2086+GARDASIL) andGroup 2 (bivalent rLP2086+saline). The proportion of subjects withresults meeting these criteria was higher after 3 vaccinations comparedwith 2 vaccinations.

These results support the evidence that the immune response to bivalentrLP2086 when coadministered with the HPV vaccine, GARDASIL, yields arobust immune response that is comparable to the immune response tobivalent rLP2086+saline.

HPV GMTs.

Table B of Example 8 presents the GMTs and the corresponding CIs foreach of the 4 HPV antigens at 1 month after Vaccination 3 for Group 1(bivalent rLP2086+GARDASIL) and Group 3 (saline+GARDASIL) in theevaluable immunogenicity population.

For Group 3, the HPV GMTs at baseline (before Vaccination 1) and at 1month after Vaccination 3 were 6.0 and 550.3, respectively, for HPV-6;4.3 and 1084.3, respectively, for HPV-11; 6.1 and 4763.4, respectively,for HPV-16; and 5.3 and 1047.4, respectively, for HPV-18. For Group 1(bivalent rLP2086+GARDASIL), the HPV GMTs at baseline (beforeVaccination 1) and at 1 month after Vaccination 3 were 5.8 and 451.8,respectively for HPV-6; 4.2 and 892.9, respectively, for HPV-11; 5.8 and3695.4, respectively, for HPV-16; and 5.2 and 744.0, respectively, forHPV-18. Overall, the GMTs were numerically higher for Group 3 comparedwith Group 1. Reverse cumulative distribution curves (RCDCs) showing thedistribution of titers for HPV-6, HPV-11, HPV-16, and HPV-18 wereassessed for Group 1 (bivalent rLP2086+GARDASIL) and Group 3(saline+GARDASIL) at all sampling time points for the evaluableimmunogenicity population. The RCDCs showed robust immune responsesamong subjects after Vaccination 3 for both Group 1 and Group 3.

Summary of Immune Response to GARDASIL.

The GMTs to HPV antigens were numerically higher for Group 3(saline+GARDASIL) as compared with Group 1 (bivalent rLP2086+GARDASIL),and the observed HPV GMTs after Vaccination 3 were indicative of arobust immune response for both groups. RCDCs also supported robustimmune responses after Vaccination 3 for both Group 1 and Group 3. Thiswas also supported by the proportion of subjects with seropositivestatus for the 4 HPV antigens, which was >99% at 1 month afterVaccination 3 for both groups. The younger age subgroup had higher HPVGMTs in Group 3 (saline+GARDASIL) than the older age subgroup. Thisdifference was maintained when GARDASIL was given concomitantly withbivalent rLP2086.

Immunogenicity Conclusions. The noninferiority criteria of bivalentrLP2086_GARDASIL compared to saline+GARDASIL or compared to bivalentrLP2086+saline required that the lower limit of the 2-sided 95% CIs forthe geometric mean titer ratios (GMRs) for antibodies to HPV for all 4HPV antigens (HPV-6, HPV-11, HPV-16, and HPV-18) and for hSBA titersusing 2 primary MnB test strains (A22 and B24) 1 month after Vaccination3 be greater than 0.67. This prespecified threshold was met for both MnBstrains and 3 of the 4 HPV antigens. For HPV-18, the lower limit of the2-sided 95% CIs for the GMR was slightly below the prespecifiedthreshold of 0.67, at 0.62.

Seroconversion for all 4 HPV antigens was achieved by 99% or more of thesubjects for the groups that received GARDASIL concomitantly withbivalent rLP2086 or with saline. The RCDCs for all 4 HPV antigens showthat the majority of subjects achieved a response above theseroconversion threshold at 1 month after Vaccination 3. Robust GMTsrelative to baseline were observed for both groups that receivedGARDASIL.

The 4-fold rise responses to 2 primary MnB test strains (A22 and B24)were similar (ranged from 83.4% to 86.4%) between the group thatreceived bivalent rLP2086+GARDASIL (85.3% and 83.4%, respectively) andthe group that received bivalent rLP2086+saline (86.4% and 84.8%,respectively).

Further descriptive analyses of the response to bivalent rLP2086 wereperformed using 4 primary MnB test strains (A22, A56, B24, and B44). Ahigh proportion of subjects achieved an hSBA titer fold rise ≥4 and thecomposite response (all 4 primary MnB test strains and the sameimmunogenicity/efficacy endpoint definition as used in the Phase 3clinical program) for the evaluable immunogenicity population for bothgroups that received bivalent rLP2086, either concomitantly withGARDASIL (bivalent rLP2086+GARDASIL) or with saline (bivalentrLP2086+saline), 1 month after Vaccination 2 or 3. These responses aresubstantially higher than an hSBA titer ≥1:4 that has been demonstratedto correlate with protection against meningococcal disease includingserogroup B disease. These results also indicate and support theevidence of a robust immune response to bivalent rLP2086 whetheradministered with saline or concomitantly with GARDASIL.

Conclusions: Data indicate that robust immune responses to both vaccineswere generated after concomitant administration of rLP2086+HPV4.Prespecified noninferiority criteria were met for 5 of 6 antigens.Although GMRs to HPV-18 narrowly missed noninferiority criteria, thehigh proportion of responders (≥99%) indicates clinical effectiveness isexpected to be maintained after concomitant administration. BivalentrLP2086 was well tolerated and elicited a robust immune response to teststrains expressing fHBPs heterologous to those in the vaccine.

TABLE A Comparison of Geometric Mean Titers at 1 Month After Vaccination3 (Evaluable Immunogenicity Population) Group 1 Group 2 Group 3 StrainrLP2086 + HPV4 rLP2086 + Saline Saline + HPV4 Ratio^(d) [Variant] n^(a)GMT^(b) (95% Cl)^(c) n^(a) GMT^(b) (95% Cl)^(c) n^(a) GMT^(b) (95%Cl)^(c) (95% Cl)^(e) HPV antigens (Group 1 vs Group 3) HPV-6 813 451.8(417.5, 489.0) 423 550.3 (490.4, 617.6) 0.82 (0.72, 0.94) HPV-11 813892.9 (839.5, 949.6) NA 423 1084.3 (997.3, 1179.0) 0.82 (0.74, 0.91) 4234763.4 (4285.9, 5294.2) 0.78 (0.68, HPV-16 813 3695.4 (3426.3, 3985.7)0.88) HPV-18 813 744.0 (687.7, 805.0) 423 1047.4 (939.0, 1168.3) 0.71(0.62, 0.81) hSBA strains (Group 1 vs Group 2) PMB80 [A22] 803 53.3(50.2, 56.7) 801 57.8 (54.4, 61.4) NA 0.92 (0.85, 1.00) 0.92 (0.84,PMB2948 [B24] 788 25.8 (24.1, 27.6) 793 28.0 (26.2, 29.9) 1.01) Cl =confidence interval; GMT = geometric mean titer; HPV = humanpapillomavirus; hSBA = serum bactericidal assay using human complement;LLOQ = lower limit of quantitation; NA = not applicable. Note: LLOQ = 11mMU/ml for HPV-6, 8 mMU/ml for HPV-11; 11 mMU/ml for HPV-16; and 10mMU/ml for HPV-18. LLOQ = 1:16 for A22; 1:8 for A56, B24, and B44.Results below the LLOQ were set to 0.5*LLOQ for analysis. ^(a)n = numberof subjects with valid and determinate assay results for the givenantigen or strain. ^(b)Geometric mean titers (GMTs) were calculatedusing all subjects with valid and determinate ssay results at 1 monthafter Vaccination 3. ^(c)Confidence intervals (Cls) are backtransformations of confidence levels based on the Student t distributionfor the mean logarithm of assay results. ^(d)Ratios of GMTs (Group1/Group 3 for HPV antigen titers and Group 1/Group 2 for hSBA straintiters). ^(e)Confidence Intervals (Cis) for the ratio are backtransformations of a confidence interval based on the Student tdistribution for the mean difference of the logarithms of the measures(Group 1-Group 3 for HPV titers and Group 1-Group 2 for hSBA straintiters).

TABLE B Comparison of Subjects Achieving HPV Seroconversion at 1 MonthAfter Vaccination 3 - Baseline HPV Seronegative Evaluable ImmunogenicityPopulation Group 1 Group 3 Seropositive rLP2086 + HPV4 Saline + HPV4Difference Antigen Criteria N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%)(95% CI)^(c) (%)^(d) (95% CI)^(e) HPV-6 ≥20 mMU/mL 802 797 (99.4) (98.6,99.8) 414 411 (99.3) (97.9, 99.9) 0.1 (−0.9, 1.5) HPV-11 ≥16 mMU/mL 801798 (99.6) (98.9, 99.9) 417 415 (99.5) (98.3, 99.9) 0.1 (−0.7, 1.3)HPV-16 ≥20 mMU/mL 800 797 (99.6) (98.9, 99.9) 413 411 (99.5) (98.3,99.9) 0.1 (−0.7, 1.3) HPV-18 ≥24 mMU/mL 805 801 (99.5) (98.7, 99.9) 418414 (99.0) (97.6, 99.7) 0.5 (−0.6, 1.9) CI = confidence interval; HPV =human papillomavirus. ^(a)N = number of subjects with baseline HPVseronegative status for the given antigen. ^(b)n = Number of subjectsachieving seroconversion (prespecified criteria) at 1 month afterVaccination 3 for the given antigen. ^(c)Exact 2-sided confidenceinterval (Clopper and Pearson) based upon the observed proportion ofsubjects. ^(d)Difference in proportions, expressed as a percentage.^(e)Exact 2-sided confidence interval (based on Chan & Zhang) for thedifference in proportions, expressed as a percentage.

Example 9: Bivalent RLP2086 Vaccine Efficacy

The efficacy of bivalent rLP2086 has been inferred using hSBA responsesas the surrogate of efficacy and demonstration of serum bactericidalantibody responses to invasive N. meningitidis serogroup B (MnB)strains.

Four MnB strains, representative of invasive meningococcal disease (IMD)causing strains, were used in the evaluation. Each MnB test strainexpresses an fHBP protein variant (A22, A56, B24 or B44) that isheterologous (differs) from the vaccine components (A05 and B01).

The efficacy of bivalent rLP2086 was assessed in 3 randomized controlledPhase II studies conducted in 4,459 adolescents aged 11 through 18 yearsof age in the US and Europe. See also Example 6. A total of 2,293received at least 1 dose of 120 μg of bivalent rLP2086 using a 0-, 2-,and 6-month vaccination schedule. Efficacy was assessed by evaluatinghSBA immune responses in subjects vaccinated with bivalent rLP2086.

Efficacy was inferred using 5 co-primary immunogenicity endpoints. For 4of the 5 co-primary endpoints, pre-specified proportions of subjects hadto achieve 4-fold rises in hSBA titer to each of the 4 MnB test strainsfollowing 3 doses of bivalent rLP2086. The fifth co-primary endpoint wasa composite endpoint requiring that a prespecified high proportion ofsubjects each respond in all 4 hSBAs with the primary MnB test strainsfollowing 3 doses of bivalent rLP2086. Immune response was also assessedbased on the proportion of subjects who achieved an hSBA titer the lowerlimit of quantitation (LLOQ) 1 month after the third dose of vaccine.LLOQ is defined as the lowest amount of the antibody in a sample thatcan be measured.

Study 1 (described in Example 7 and Example 8) was a Phase II,randomized, active-controlled, observer-blinded, multicenter trial inwhich 2,499 US subjects, 11 through 17 years of age, were randomlyassigned (in a 2:2:1 ratio) to 1 of 3 groups: Group 1 received bivalentrLP2086+HPV4, Group 2 received bivalent rLP2086+Saline, and Group 3received Saline+HPV4. All vaccinations were administered on a 0-, 2-,and 6-month schedule.

Study 2 (described in Example 4) was a Phase II, randomized,placebo-controlled, single-blind trial in which 753 European subjects,11 through 18 years of age, were randomly assigned in a 1:1 ratio to 2groups: Group 1 received bivalent rLP2086 at 0-, 2-, and 6-months anddTaP-IPV (diphtheria, tetanus, acellular pertussis-inactivated poliovirus) at Month 0. Group 2 received Saline at 0-, 2-, and 6-months anddTaP-IPV at Month 0.

Study 3 (described in Example 5) was a Phase II, randomized,placebo-controlled, single-blind, multicenter trial in which 1,713European subjects, 11 through 18 years of age, were randomly assigned ina 3:3:3:2:1 ratio to 5 groups. Subjects received 2 or 3 doses ofbivalent rLP2086 administered on a 0-, 1-, and 6-month schedule (Group1); on a 0-, 2-, and 6-month schedule (Group 2); on a 0- and 6-monthschedule (Group 3); on a 0- and 2-month schedule (Group 4); or on a 0-and 4-month schedule (Group 5). Saline injections (1 or 2 dosesdepending on group) were administered in each group to maintain theblind.

Results in Studies 1, 2, and 3 among subjects who received a 3-doseseries of bivalent rLP2086 at 0-, 2-, and 6-months are described abovein the respective Examples 4-8. Evaluation of the 4-fold and compositeresponse rates were exploratory endpoints for all studies. The 4-foldresponse rates showed that the lower bounds of the 95% ConfidenceInterval (CI) for all 4 endpoints were similar among the 3 studies andconsistently met the threshold limits for the Phase III endpoints. Theproportion of subjects achieving hSBA titer ≥LLOQ was similar across the3 studies.

Based on the hSBA data acquired following 2 administrations of thevaccine given 1 or 2 months apart, 2 doses of vaccine administered overthese intervals may provide protection to individuals at increased risk,due to potential exposure to a case of meningococcal serogroup Bdisease. The responses observed after 2 vaccine administrationsdelivered 1 or 2 months apart showed that a proportion of subjectsexpressed hSBA levels equal to or above the LLOQ values for each of the4 primary test strains (see Study 1 results for Group 1 and Group 2; seeStudy 2 results for Group 1; see Study 3 results for Group 2). A thirddose of the vaccine, administered at 6 months, can achievevaccine-mediated protection.

Concomitant Vaccine Administration.

Study 1 (described in Example 7 and Example 8) evaluated the concomitantuse of bivalent rLP2086 and HPV4 in US adolescents. The study endpointsincluded noninferiority assessment of the immune response for the fourHPV4 antigens (based on geometric mean titer [GMT]) and for bivalentrLP2086 (based on hSBA using two MnB test strains [variants A22 andB24]) 1 month after the third vaccination. HPV4 immune response was alsoevaluated by seroconversion for each of the 4 HPV antigens.

Study 1 shows the comparison of the geometric mean titers (GMTs) of theantibodies to HPV antigens for Group 1 (bivalent rLP2086+HPV4) and Group3 (Saline+HPV4), with their corresponding GMT ratio (GMRs) between Group1 and Group 3 and the 2-sided 95% CIs of the ratios. Study 1 alsoprovides the comparison of hSBA GMTs to the 2 primary MnB test strainsfor Group 1 and Group 2 with their corresponding GMRs between Group 1and Group 2 and the 2-sided 95% CI of the ratios. The criterion fornoninferiority margin was 1.5-fold, which corresponds to a value of 0.67for the lower limit of the 2-sided 95% CI of the GMR. The 1.5-foldcriterion of 0.67 was met for all the MnB test strains and the HPVantigens except for HPV-18, which had a lower bound 95% confidenceinterval (CI) of 0.62. Although the response to HPV-18 did not meet thepre-specified noninferiority criterion, the difference was marginal. Ina separate analysis, ≥99%, of subjects seroconverted to all 4 HPVantigens in both the Saline+HPV4 and bivalent rLP2086+HPV4 groups.

Example 10: Bivalent rLP2086 Elicits Antibodies in Individuals thatProvide Broad Coverage Against MnB Strains Expressing Prevalent andOutbreak-Associated fHBP Variants

Bactericidal antibodies measured in serum bactericidal assays usinghuman complement (hSBAs) have been correlated with protection frommeningococcal disease and hSBA responses have been used routinely assurrogates of vaccine efficacy. Global epidemiological studies of fHBPdiversity revealed that ˜80% of meningococcal disease is caused bystrains that express one of 10 prevalent fHBP variants.

Methods:

hSBA responses to Neisseria meningitidis serogroup B (MnB) strainsexpressing the 10 most prevalent fHBP variants in the US and Europe(B24, B16, B44, A22, B03, B09, A12, A19, A05 and A07) in individualhuman subjects immunized with bivalent rLP2086 were evaluated. MnBstrains expressing these ten most prevalent variants represent thebreadth of fHBP diversity, including 5 of the 6 major fHBP subgroups,that are representative of >98% and 97% of strains (by subgroup) in theMnB SBA strain pool, and US subset of the MnB SBA strain pool,respectively. Twenty-three MnB test strains were obtained from Pfizer'sMnB SBA strain pool (N=1263) that represent strains systematicallycollected from the US and Europe between the years 2000 and 2006. Inaddition, isolates from recent MnB disease outbreaks were included inthe analysis. Matched prevaccination and postvaccination sera (postdose2 and postdose 3) were obtained randomly from adolescent and young adultsubjects enrolled in clinical studies B1971005, B1971012 or B1971003.

To provide additional information supporting the potential coverageafforded by vaccination with bivalent rLP2086, hSBAs were performed withthe outbreak strains and serum samples from nine subjects immunized withbivalent rLP2086 (clinical study B1971012, described in Example 5 andExample 6. The subjects (11 to <19 years of age) had received 3 doses ofbivalent rLP2086 at 0, 2 and 6 months. To ensure a conservative hSBAassessment the nine subjects were selected in a non-biased manner from aset of subjects with no baseline hSBA activity against the primary MnBtest strains. Two of the clonal Princeton University outbreak strains(PMB5021 and PMB5025) and two of the UCSB outbreak strains (one fromeach of the two genetic clusters, PMB4478 and PMB4479, were tested.

Genetic characterization of the clonal Princeton University MnB OutbreakStrains is as follows: data suggest that the Princeton Universityoutbreak strains are clonal. Each of the strains was typed as CC41/44(ST 409) and expressed fHBP variant B153 (SEQ ID NO: 6). The strains hadidentical allele assignments for NHBA (2), porA (subtype P1.5-1, 2-2)and porB (3-82), all were null for nadA, and all had the same pulsedfield gel electrophoresis (PFGE) profile (429).

Genetic characterization of the 2013 University of California SantaBarbara Outbreak Strains is as follows: The UCSB strains were typed asCC32(ET5; ST32), expressed fHBP variant B24, and are related to theOregon clone that has been associated with hyperendemic serogroup Bdisease since 1993. Unlike the Princeton outbreak group of strains, theUCSB strains segregated genetically into two distinct clusters that weredifferentiated by their PFGE profile (468 or 467) and porB type (3-461or 3-24). The strains had identical allele assignments for NadA (1),NHBA (5), porA (subtype P1.7, 16-20)

hSBA titers at baseline for all subjects and all outbreak strains were<4, indicating that the subjects had no protective antibodies to any ofthe outbreak strains prior to immunization with bivalent rLP2086.

Results:

All 23 MnB strains were susceptible in hSBA with sera from individualsubjects immunized with bivalent rLP2086. Strains representing all 10prevalent fHBP variants as well as additional strains were all killed byhSBA. Baseline hSBA seroprotection rates (proportions of subjectsachieving hSBA titers ≥1:4) were generally low. The lower seroprotectiverates observed in subjects before immunization with bivalent rLP2086exemplify the vulnerability of a non-vaccinated adolescent or youngadult population to MnB disease. However, robust seroprotection rateswere observed in adolescents and young adults with postvaccination sera:seroprotection rates >70% were observed for 83% of these strainsdepending on MnB strains and population tested. Postvaccinationseroprotection rates for strains expressing the most prevalent subfamilyA and B fHBP variants, B24 and A22, ranged from 81.0% to 100%, and 77.8%to 100% for recent outbreak strains expressing fHBP variants B24 andB153. Furthermore, robust postdose 2 responses (compared to baseline) toall outbreak strains were observed in these subjects, ranging from 56 to89% depending on the outbreak strain used in the hSBA. In contrast,prevaccination seroprotective rates were low, or not detectable, forrecent US outbreak strains. The hSBA responses to the PrincetonUniversity and UCSB outbreak strains are shown in FIG. 2.

Conclusions: Bivalent rLP2086 elicits robust seroprotective hSBAresponses in individuals to diverse invasive MnB strains expressingprevalent fHBPs in the US and Europe, as well as newly emerging variants(B153)(SEQ ID NO: 6). The proportion of subjects that showed aseroprotective response after immunization with bivalent rLP2086 greatlyexceeded the proportion of subjects that was seroprotected at baseline.The data support that bivalent rLP2086 has the potential to providebroad protection of adolescents and young adults from invasivemeningococcal serogroup B disease, including disease from recentoutbreaks.

>B153  (SEQ ID NO: 6)CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFDKLPKGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGGKAEEVAGSAEVKTVNGIRHIGLAAKQ

Example 11: Immunogenicity and Safety of Bivalent rLP2086, aMeningococcal Serogroup B Vaccine, Coadministered with Tdap and MCV4 inUS Adolescents

A Phase 2, Randomized, Active-Controlled, Observer Blinded Trial, toAssess the Safety, Tolerability, and Immunogenicity of MCV4, TdapVaccine and Bivalent rLP2086 Vaccine When Administered Concomitantly inHealthy Subjects Aged 0 to <13 Years

Background:

Concurrent administration of bivalent rLP2086 with vaccines recommendedin the US may improve adherence to vaccine schedules.

Objective:

To determine if immune responses induced by coadministration ofMENACTRA® [meningococcal A, C, Y and W-135 polysaccharide conjugatevaccine (MCV4)] and ADACEL® [tetanus toxoid, reduced diphtheria toxoid,acellular pertussis vaccine (Tdap)] with TRUMENBA® [meningococcal B(MnB) vaccine], approved in the United States (bivalent rLP2086)] werenoninferior to MCV4+Tdap or bivalent rLP2086 alone

Design/Methods:

Healthy adolescents aged ≥10 to <13 y received

MCV4+Tdap+bivalent rLP2086, MCV4+Tdap, or bivalent rLP2086. BivalentrLP2086 immunogenicity was assessed by serum bactericidal assay usinghuman complement (hSBA) with 2 MnB test strains expressingvaccine-heterologous fHBP variants; multiplexed LUMINEX assays and/orrabbit SBAs assessed immunogenicity of MCV4/Tdap antigens. Safety wasassessed.

Results:

2648 subjects received MCV4+Tdap+bivalent rLP2086, MCV4+Tdap, orbivalent rLP2086. Immune responses to MCV4, Tdap, and bivalent rLP2086given concomitantly were noninferior to immune responses to MCV4+Tdap orbivalent rLP2086 alone. 62.3-68.0% and 87.5-90% of MCV4+Tdap+bivalentrLP2086 recipients after vaccination 2 and 3, respectively, hadprespecified seroprotective hSBA titers to 2 MnB test strains; bivalentrLP2086 alone induced similar responses. Concomitant administration didnot significantly increase local reactions or systemic events comparedto bivalent rLP2086 alone.

Bivalent rLP2086 given concomitantly with MCV4+Tdap met allnoninferiority immunogenicity criteria without a clinically significantincrease in reactogenicity. Convenience associated with concomitantadministration may improve adherence to recommended schedules.

This study (B1971015) assessed the immunogenicity of quadrivalentmeningococcal polysaccharide conjugate (MCV4) vaccine; tetanus,diphtheria, and acellular pertussis (Tdap) vaccine; and bivalent rLP2086when administered concomitantly. It also assessed the safety,tolerability and immunogenicity of bivalent rLP2086 in healthy subjectsaged ≥10 to <13 years in the United States. The 2 MnB test strains usedin hSBA to evaluate noninferiority of the immune response to bivalentrLP2086 when given with or without the licensed vaccines express the twomost prevalent fHBPs in the United States, namely A22 and B24.

Saline was used as a placebo to maintain the study blind because therewas no proven safe, immunogenic, and effective vaccine against MnB thatcould serve as an active control. In addition, saline was chosen as aninert control as opposed to a control vaccine with excipients (such asaluminum) because the safety objective to describe the tolerabilityprofile of the investigational product was met with saline rather thananother vaccine.

Approximately 2625 subjects were to participate in this study atapproximately 88 sites in the United States (approximately 30 subjectsat each site). Subjects were randomly assigned to 1 of 3 groups in a1:1:1 ratio (Group 1: Group 2: Group 3) (Table A). Subjects receivedinvestigational products at each of the vaccination visits (Visits 1, 3,and 5).

Subjects presenting with any of the following were not to be included inthe study: Previous vaccination with any meningococcal serogroup Bvaccine; Vaccination with any diphtheria, tetanus or pertussis vaccinewithin 5 years of the first study vaccination; Previous vaccination withany MCV4 vaccine.

TABLE A Study Design Approx Month 0 1 2 3 6 7 12 Visit 1 Visit 2 Visit 3Visit 4 Visit 5 Visit 6 Visit 7 Vaccination MCV4 + Tdap + rLP2086rLP2086 Final (Group 1) rLP2086 telephone contact Vaccination MCV4 +Tdap + Saline Saline Final (Group 2) Saline telephone contactVaccination Saline + Saline + rLP2086 rLP2086 MCV4 + Final (Group 3)^(a)rLP2086 Tdap^(b) telephone contact Blood draw ~20 mL ~20 mL ~20 mL ~20mL (all groups) Abbreviation: rLP2086 = bivalent recombinant lipoprotein2086 vaccine. ^(a)Subjects in Group 3 who withdrew or discontinued earlyshould have been contacted to return to clinic to receive MCV4 and Tdapvaccinations. ^(b)Study sites were able to determine whether a subjectwas in Group 3 or not at Visit 6 (1 month after the last dose of eithersaline or rLP2086) in order to identify individuals who required MCV4 +Tdap. Subject assignment to Group 1 and 2 remained blinded throughoutthe study.

During the study, subjects in Groups 1 and 3 received bivalent rLP2086at approximately 0, 2, and 6 months (Visits 1, 3, and 5, respectively)in the left arm. For subjects in Group 2, saline was administered byintramuscular (IM) injection (0.5 mL) at approximately 0, 2, and 6months (Visits 1, 3, and 5, respectively) in the left arm. Subjects inGroup 3 received 2 IM saline injections at 0 months in the right arm.MCV4 and Tdap vaccines were administered once only by IM injection at 0months (Groups 1 and 2) in the right arm. Following the last blood drawat Visit 6 (post-Vaccination 3 blood draw), subjects in Group 3 receivedthe MCV4 and Tdap vaccines.

MCV4 and Tdap vaccines were administered once only by IM injection at 0months (Groups 1 and 2) into the upper deltoid muscle of the right arm.The vaccines were administered at least 5 cm apart. Subjects in Group 3received MCV4 and Tdap vaccines at Visit 6 following their last blooddraw.

Blood samples (approximately 20 ml each) were collected immediatelybefore Vaccination 1, 28 to 41 days after Vaccination 1, 28 to 42 daysafter Vaccination 2, and 28 to 42 days after Vaccination 3 forimmunogenicity analyses.

The bivalent rLP2086 vaccine used was in a 0.5 mL dose formulated tocontain 60 μg each of a purified subfamily A rLP2086 protein (variantA05, SEQ ID NO: 1) and a purified subfamily B rLP2086 protein (variantB01, SEQ ID NO: 2), 0.15 M sodium chloride, 2.8 molar ratio polysorbate80, and 0.25 mg of Al³+ as aluminum phosphate (AlPO4) as a stabilizer in10 mM histidine-buffered saline at pH 6.0.

The MCV4 vaccine was MENACTRA® [meningococcal (Groups A, C, Y and W-135)polysaccharide diphtheria toxoid conjugate vaccine (MCV4)], acommercially available vaccine. MENACTRA is a solution supplied as a 0.5mL dose for IM injection. The vaccine contains N. meningitidis serogroupA, C, Y and W-135 capsular polysaccharide antigens individuallyconjugated to diphtheria toxoid protein. The four meningococcalcomponents, present as individual serogroup-specific glycoconjugates,compose the final formulated vaccine. No preservative or adjuvant isadded during manufacture. Each 0.5 mL dose could contain residualamounts of formaldehyde of less than 2.66 μg (0.000532%), bycalculation. MENACTRA is manufactured as a sterile, clear to slightlyturbid liquid. Each 0.5 mL dose of vaccine is formulated in sodiumphosphate buffered isotonic sodium chloride solution to contain 4 μgeach of meningococcal A, C, Y and W-135 polysaccharides conjugated toapproximately 48 of diphtheria toxoid protein carrier.

The Tdap vaccine was ADACEL® (tetanus toxoid, reduced diphtheria toxoidand acellular pertussis vaccine adsorbed [Tdap]), a commerciallyavailable vaccine. ADACEL is supplied as a 0.5 mL dose for IM injection.The vaccine is a sterile isotonic suspension of tetanus and diphtheriatoxoids and pertussis antigens adsorbed on aluminum phosphate. Each 0.5mL dose contains 5 Lf tetanus toxoid, 2 Lf diphtheria toxoid, andacellular pertussis antigens (2.5 μg detoxified pertussis toxin, 5 μgfilamentous hemagglutinin, 3 μg pertactin [PRN], 5 μg fimbriae types 2and 3). Other ingredients per 0.5 mL dose include 1.5 mg aluminumphosphate (0.33 mg aluminum) as the adjuvant, ≤5 μg residualformaldehyde, <50 ng residual glutaraldehyde and 3.3 mg (0.6% v/v)2-phenoxyethanol (not as a preservative). ADACEL does not contain apreservative.

Sterile normal saline solution for injection (0.9% sodium chloride) wasin a 0.5 mL dose.

Bivalent rLP2086 Serum Bactericidal Assays.

For assessment of the immune response to bivalent rLP2086, functionalantibodies were analyzed in validated hSBAs. The hSBA measuresantibodies in human sera that result in complement mediated killing ofthe target MnB strain. Sera obtained from all subjects in Groups 1 and3, prior to the first vaccination with bivalent rLP2086, approximately 1month after the second vaccination with bivalent rLP2086 (Visit 3), andapproximately 1 month after the third vaccination with bivalent rLP2086(Visit 5), were tested in hSBAs with strains PMB80 [A22] and PMB2948[B24].

MCV4 Concomitant Vaccine SBAs.

For assessment of the immune response to the MCV4 vaccine, functionalantibodies were analyzed in serum bactericidal assays using rabbitcomplement (rSBAs) with meningococcal strains representing serogroups A,C, Y and W-135. Sera obtained from all subjects in Groups 1 and 2 priorto the first vaccination with MCV4 (Visit 1) and 1 month aftervaccination with MCV4 (Visit 2) were used in these assays.

Antigen Detection Assays for MCV4 and Other Concomitant Vaccines.

Assessments of diphtheria, tetanus, and pertussis antibody responseswere performed using a validated and multiplexed LUMINEX multiplex assay(MCV4 LXA). Sera obtained from all subjects in Groups 1 and 2 prior tothe first vaccination with MCV4 and Tdap (Visit 1) and 1 month aftervaccination with MCV4 and Tdap (Visit 2) were used in the assay.Additional assessments of meningococcal serogroups A, C, Y and W-135responses were performed using a validated multiplexed LXA (A,C,Y,WLXA). Sera obtained from all subjects in Groups 1 and 2 prior to thefirst vaccination with MCV4 and Tdap (Visit 1) and 1 month aftervaccination with MCV4 and Tdap (Visit 2) were used in these assays.

Sample Size.

The antibody responses to the 10 antigens in the marketed vaccines and 2MnB strains were assumed to be independent. The criteria for thenoninferiority margin were 1.5-fold.

Methods of Analysis.

The LLOQ for PMB80 (A22) was 1:16 and the LLOQ for PMB2948 (B24) was1:8. The LLOQs for Tdap/MCV4 assays are listed in Table B.

TABLE B LLOQs for Assays to determine immune responses to MarketedVaccine Antigens Vaccine-Assay/Antigen LLOQ MCV4-rSBA Meningococcalserogroup A 1:32 Meningococcal serogroup C 1:64 Meningococcal serogroupY 1:128 Meningococcal serogroup W-135 1:64 MCV4-LXA (IgG) Meningococcalserogroup A 0.1795 ug/mL^(a) Meningococcal serogroup C 0.0940 ug/mL^(b)Meningococcal serogroup Y 0.2485 ug/mL^(c) Meningococcal serogroup W-1350.2530 ug/mL^(d) Tdap-LXA Diphtheria 0.037 IU/ml Tetanus 0.05 IU/mlPertussis toxoid 0.9 IU/ml Pertussis filamentous haemagglutinin 2.9IU/ml Pertussis pertactin 3.0 IU/ml Pertussis fimbriae types 2 + 3 10.6IU/ml Abbreviations: IgG = immunoglobulin G; LLOQ = lower limit ofquantitation; LXA = LUMINEX assay; MCV4 = quadrivalent meningococcalpolysaccharide conjugate; rSBA = serum bactericidal assay using rabbitcomplement; Tdap = tetanus, diphtheria, and acellular pertussis.^(a)Postvalidation LLOQ presented in table; validation LLOQ was 0.1037μg/mL. ^(b)Postvalidation LLOQ presented in table; validation LLOQ was0.0204 μg/mL. ^(c)Postvalidation LLOQ presented in table; validationLLOQ was 0.1104 μg/mL. ^(d)Postvalidation LLOQ presented in table;validation LLOQ was 0.1198 μg/mL.

Analysis for Coprimary Objectives (Noninferiority Objectives) GMTs andGMCs.

For MnB strains PMB80 (A22) and PMB2948 (B24), hSBA titers measured ateach blood sampling time points (Visits 1, 4, and 6) werelogarithmically transformed for analysis and GMTs were computed for eachprimary strain and for each group at each blood sampling time pointalong with 2-sided 95% confidence intervals (CIs). The CIs wereconstructed by back transformation of the confidence limits computed forthe mean of the logarithmically transformed assay data based onStudent's t distribution. The GMTs determined in rSBAs to each of the 4MCV4 antigens and the GMCs determined in LXAsr to each of the 4 MCV4antigens were summarized similarly at Visit 1 and Visit 2. The GMCdetermined by LXA to each of the 6 antigens in the Tdap vaccine weresummarized at Visit 1 and Visit 2.

Geometric Mean Ratios.

For the purpose of testing the noninferiority hypotheses, geometric meanratios (GMRs) between corresponding groups at each applicable visit(Group 1/Group 3 for the hSBA titer at Visit 6 and Group 1/Group 2 forthe MCV4/Tdap antigens at Visit 2), along with 95% CI were used. Themean difference of the logarithmically transformed results wasequivalent to the mean of the ratio on the logarithmic scale: log (Group1/Group 2)=log(Group 1)−log (Group 2). The proper exponentialtransformation was used to calculate the GMR. Similarly, the 2-sided,95% CIs for the ratios were constructed by back transformation of theCIs for the mean difference of the logarithmically transformed assayresults computed using Student's t distribution. Statistical inferencewas based on the CIs of the GMRs (ie, lower bound of the 95% CI was>0.67).

Noninferiority for Marketed Vaccine (Coprimary for MCV4/Tdap).

For the first coprimary objective, antibody titers or concentrations toeach of the 10 antigens in the marketed vaccines measured at 1 monthafter vaccination (Visit 2) were logarithmically transformed foranalysis and GMs were computed for Group 1 and Group 2. The 2-sided 95%CI of the ratio ([bivalent rLP2086+MCV4/Tdap]/[MCV4/Tdap]) of GMs, wereprovided for each of the 10 antigens.

Noninferiority for rLP2086 Vaccine (Coprimary for Bivalent rLP2086).

For the second coprimary objective, the hSBA titers for each of the 2primary strains measured at Visit 6 were logarithmically transformed foranalysis and GMTs were computed for Group 1 and Group 3. The 2-sided 95%CI of the GMRs ([bivalent rLP2086+MCV4/Tdap]/bivalent rLP2086) wereprovided for each of the 2 primary strains. The study coprimaryobjectives on noninferiority of both marketed vaccines (MCV4/Tdap) andinvestigational product (bivalent rLP2086) was achieved when the lowerlimit of the 2-sided 95% CI for the GMRs at Visit 2 for ([bivalentrLP2086+MCV4/Tdap]/[MCV4/Tdap], and at Visit 6 for [bivalentrLP2086+MCV4/Tdap]/bivalent rLP2086), were greater than 0.67 for all 10MCV4/Tdap antigens and each of the 2 primary strains. GMRs (Group1/Group2) were descriptively summarized with 2-sided 95% CI, for IgGconcentrations to each of the 4 antigens in MCV4 at Visit 2.

Analysis of hSBA Data.

The number and proportions of subjects who achieved hSBA titers ωLLOQ,≥1:4, ≥1:8, ≥1:16, ≥1:32, ≥1:64, and ≥1:128 at each blood sampling timepoint, and with at least 2-fold, 3-fold, and 4-fold rise in hSBA titerfrom baseline to after Vaccination 2 and after Vaccination 3, weredescriptively summarized along with the exact 2-sided 95% CI (orClopper-Pearson confidence limit) of the proportion for the primary MnBtest strains PMB80 (A22) and PMB2948 (B24) for Group 1 and Group 3. Theexact CI for the proportion was computed using the F distribution. As anadditional analysis for noninferiority of bivalent rLP2086, the hSBA4-fold response rate for the A22 variant (PMB80) and B24 variant(PMB2948) were compared between Group 1 and Group 3. The difference ofthe rates (bivalent rLP2086/MCV4/Tdap-bivalent rLP2086 or Group 1-Group3) and exact 2-sided 95% CIs on the difference were provided. The exactCIs were computed using the standardized test statistic andgamma=0.000001. This was considered a descriptive analysis.

Analysis of MCV4/Tdap Immunogenicity.

The proportion of subjects who achieved a seroresponse to each MCV4/Tdapantigen, and the proportion of subjects who achieved an antibody level≥1.0 IU/mL for tetanus and diphtheria toxoids were summarized with exact2-sided 95% CIs for Group 1 and Group 2. Noninferiority with respect toseroresponse rates to each MCV4/Tdap antigen and the proportion ofsubjects achieving an antibody level ≥1.0 IU/mL for tetanus anddiphtheria toxoid (Group 1-Group 2) were analyzed.

Immune Response to Bivalent rLP2086 and MCV4/Tdap Antigens whenAdministered Concomitantly. Noninferiority Analyses of Geometric MeanConcentrations and Geometric Mean Titers to Tdap and MCV4 Antigens.

The first coprimary objective of this study was to demonstrate that theimmune response based on GMTs (or GMCs where applicable) induced by MCV4and Tdap vaccines given with bivalent rLP2086 (Group 1) was noninferiorto the immune response induced by MCV4 and Tdap vaccines alone (Group 2)when measured 1 month after the first vaccination (Visit 2) in bothgroups. The primary endpoints for the first coprimary objective were theGMTs or GMCs to each of the 10 antigens in the marketed vaccines at 1month after the first vaccination (Visit 2), among subjects in Groups 1and 2.

The comparison of GMTs (or GMCs where applicable) to the 10 MCV4 andTdap antigens for Group 1 and Group 2, with their corresponding GMT (orGMC where applicable) ratios (GMRs) of Group 1 to Group 2 and the2-sided 95% CI of the ratios, is presented in Table C. The criterion forthe noninferiority margin was 1.5-fold, which corresponds to a value of0.67 for the lower limit of the 2-sided 95% CI of the GMR.

For the evaluable immunogenicity population, GMRs of Group 1 to Group 2at 1 month after Vaccination 1 for the Tdap antigens were 0.94 fordiphtheria toxoid (95% CI: 0.86, 1.03), 0.92 for tetanus toxoid (95% CI:0.85, 0.99), 0.93 for pertussis toxoid (95% CI: 0.85, 1.02), 0.91 forpertussis filamentous hemagglutinin (95% CI: 0.84, 0.98), 0.88 forpertussis pertactin (95% CI: 0.80, 0.98), 0.90 for pertussis fimbrialagglutinogens types 2+3 (95% CI: 0.74, 1.08); and the GMRs for the MCV4antigens were 0.91 for serogroup A (95% CI: 0.82, 1.01), 1.02 forserogroup C (95% CI: 0.90, 1.15), 0.99 for serogroup Y (95% CI: 0.89,1.09), and 0.93 for serogroup W-135 (95% CI: 0.83, 1.04). Therefore, thelower limits of the 2-sided 95% CIs for MCV4 and Tdap antigen GMRs forGroup 1 compared to Group 2 were 0.86 for diphtheria toxoid, 0.85 fortetanus toxoid, 0.85 for pertussis toxoid, 0.84 for pertussisfilamentous hemagglutinin, 0.80 for pertussis pertactin, 0.74 forpertussis fimbrial agglutinogens types 2+3, 0.82 for serogroup A, 0.90for serogroup C, 0.89 for serogroup Y, and 0.83 for serogroup W-135. The1.5-fold criterion of 0.67 (the lower limit of the 2-sided 95% CI of theGMR) was met for all MCV4 and Tdap antigens. Therefore, the firstcoprimary objective was met.

The immune response to the Tdap+MCV4 antigens when these vaccines aregiven concomitantly with bivalent rLP2086 was noninferior to the immuneresponse obtained when they were given alone.

TABLE C Primary Immunogenicity Analysis - Comparison of Geometric Meansat 1 Month After Last Vaccination - Evaluable Immunogenicity PopulationsVaccine Group (as Randomized) Group 1 Group 2 Group 3 Antigen/StrainMCV4 + Tdap + rLP2086 MCV4 + Tdap + Saline Saline + Saline + rLP2086(Variant) N^(a) GM^(b) (95% CI)^(c) N^(a) GM^(b) (95% CI)^(c) N^(a)GM^(b) (95% CI)^(c) Ratio^(d) (95% CI)^(e) Tdap antigens Diphtheria 778  9.3 (8.67, 9.92) 780   9.8 (9.23, 10.51) N/A N/A N/A 0.94 (0.86, 1.03)Tetanus 778   9.4 (8.95, 9.98) 780  10.3 (9.75, 10.85) N/A N/A N/A 0.92(0.85, 0.99) Pertussis toxoid 778  13.2 (12.35, 14.14) 780  14.2 (13.28,15.20) N/A N/A N/A 0.93 (0.85, 1.02) Pertussis filamentous 778  112.0(106.15, 118.14) 780  122.9 (116.42, 129.84) N/A N/A N/A 0.91 (0.84,0.98) hemagglutinin Pertussis pertactin 778  202.0 (187.77, 217.25) 780 228.9 (212.72, 246.35) N/A N/A N/A 0.88 (0.80, 0.98) Pertussis fimbriae778  138.1 (121.20, 157.33) 780  154.2 (135.30, 175.79) N/A N/A N/A 0.90(0.74, 1.08) agglutinogens types 2 + 3 rSBA MCV4 antigens Serogroup A763 4647.3 (4317.66, 5002.09) 772 5113.0 (4748.73, 5505.17) N/A N/A N/A0.91 (0.82, 1.01) Serogroup C 768 1679.2 (1539.63, 1831.38) 767 1650.2(1519.01, 1792.65) N/A N/A N/A 1.02 (0.90, 1.15) Serogroup Y 771 2212.6(2056.08, 2381.08) 770 2244 9 (2088.70, 2412.89) N/A N/A N/A 0.99 (0.89,1.09) Serogroup W-135 751 5925.1 (5469.77, 6418.33) 765 6367.9 (5872.68,6904.88) N/A N/A N/A 0.93 (0.83, 1.04) hSBA MnB strains PMB80 (A22) 679 45.9 (42.74, 49.35) N/A N/A N/A 674 49.7 (46.43, 53.30) 0.92 (0.84,1.02) PMB2948 (B24) 670  24.8 (23.11, 26.58) N/A N/A N/A 656 27.4(25.58, 29.41) 0.90 (0.82, 1.00) Abbreviations: ELISA = enzyme-linkedimmunosorbent assay; EU = ELISA units; GM = geometric mean; hSBA = serumbactericidal assay using human complement; LLOQ = lower limit ofquantitation; MCV4 = quadrivalent meningococcal polysaccharide conjugate(vaccine); MnB = Neisseria meningitidis serogroup B; N/A = notapplicable; rSBA = serum bactericidal assay using rabbit complement;Tdap = tetanus, diphtheriam, and acellular pertussis (vaccine). Note: Inorder for the primary study hypothesis to be a success, the lower boundof the 95% CI of GM ratios for all Tdap/MCV4 antigens and MnB strainsmust be >0.67. Note: For Tdap and MCV4 antigens, the post-Vaccination 1evaluable immunogenicity population was used; for the MnB strains, thepost-Vaccination 3 immunogenicity population was used. The Tdap/MCV4antigens were not tested for Group 3 subjects and the MnB strains werenot tested for Group 2 subjects. Note: LLOQ = 0.037 IU/mL fordiphtheria; 0.05 IU/mL for tetanus; 0.9 EU/mL for pertussis toxoid; 2.9EU/mL for pertussis filamentous hemagglutinin; 3.0 EU/mL for pertussispertactin; 10.6 EU/mL for pertussis fimbrial agglutinogens types 2 + 3.LLOQ = 1:32 for MCV4 serogroup A, 1:64 for serogroup C, 1:128 forserogroup Y, and 1:64 for serogroup W-135. LLOQ = 1:16 for A22; 1:8 forB24. Results below the LLOQ were set to 0.5 × LLOQ for analysis. ^(a)N =number of subjects with valid and determinate assay results for thegiven antigen or strain. ^(b)GMs were calculated using all subjects withvalid and determinate assay results at 1 month after last vaccination(Visit 6 for hSBA results and Visit 2 for all other results).^(c)Confidence Intervals (CIs) are back transformations of confidencelevels based on Student t distribution for the mean logarithm of assayresults. ^(d)Ratios of GMs (Group 1/Group 2 for Tdap/MCV4 antigens andGroup 1/Group 3 for MnB strains). ^(e)CIs for the ratio are backtransformations of a confidence interval based on the Student tdistribution for the mean difference of the logarithms of the measures(Group 1 - Group 2 for Tdap/MCV4 antigens and Group 1 - Group 3 for MnBstrains).Noninferiority Analysis for hSBA GMTs to Bivalent rLP2086.

The second coprimary objective of this study was to demonstrate that thehSBA response to primary strains PMB80 (A22) and PMB2948 (B24) (based onGMTs) induced by MCV4+Tdap+bivalent rLP2086 (Group 1) was noninferior tothe immune response induced by the saline+saline+bivalent rLP2086 (Group3). The hSBA was performed 1 month after the third vaccination (Visit 6)with bivalent rLP2086 for both groups. The primary endpoints for thesecond coprimary objective were the evaluation of hSBA GMTs to each ofthe 2 primary strains (PMB80 [A22] and PMB2948 [B24]) measured at 1month after the third dose of bivalent rLP2086 (Visit 6), which wasadministered to subjects in Group 1 (MCV4+Tdap+bivalent rLP2086) andGroup 3 (saline+saline+bivalent rLP2086).

Table C presents the comparison of hSBA GMTs to the 2 primary MnB teststrains for Group 1 and Group 3 with their corresponding GMRs of Group 1to Group 3 and the 2-sided 95% CIs of the ratios, for the evaluableimmunogenicity population. The criterion for the noninferiority marginwas 1.5-fold, which corresponds to a value of 0.67 for the lower limitof the 2-sided 95% CI of the GMR.

For the evaluable immunogenicity population, the hSBA GMTs to the 2primary MnB strains at 1 month after Vaccination 3 (bivalent rLP2086)for Group 1 and Group 3 were as follows: 45.9 and 49.7 respectively forPMB80 (A22) and 24.8 and 27.4, respectively for PMB2948 (B24). The GMRsof the MCV4+Tdap+bivalent rLP2086 group to the saline+saline+bivalentrLP2086 group at 1 month after Vaccination 3 (bivalent rLP2086) were0.92 for PMB80 (A22) (95% CI: 0.84, 1.02) and 0.90 for PMB2948 (B24)(95% CI: 0.82, 1.00). The lower limits of the 2-sided 95% CIs for thehSBA GMRs for Group 1 compared to Group 3 were 0.84 for PMB80 (A22) and0.82 for PMB2948 (B24), which are both greater than 0.67 and thenoninferiority margin of 1.5-fold was met. Therefore, the secondcoprimary objective was achieved.

The immune response to the bivalent rLP2086 vaccine when givenconcomitantly with Tdap+MCV4 was noninferior to the immune responseobtained when the vaccine was given alone.

Noninferiority Analysis for the Coprimary Objectives.

The first coprimary objective for the noninferiority of bivalent rLP2086given with MCV4 and Tdap vaccines compared to both MCV4 and Tdapvaccines alone was tested by examining a noninferiority margin set at1.5 fold, which corresponds to a value of 0.67 for the lower limit ofthe 2-sided 95% CI of the GMR for each of the 10 MCV4 and Tdap antigensfor the post-Vaccination 1 evaluable immunogenicity population. Thiscriterion was met for all MCV4 and Tdap antigens tested.

The second coprimary objective for the noninferiority of bivalentrLP2086 given with MCV4 and Tdap vaccines compared to the bivalentrLP2086 alone was tested by examining a noninferiority margin set at 1.5fold, which corresponds to a value of 0.67 for the lower limit of the2-sided 95% CI of the GMR for each of the 2 primary MnB strains(variants A22 and B24) in the post-Vaccination 3 evaluableimmunogenicity population when measured 1 month after Vaccination 3.This criterion was met for both MnB test strains PMB80 (A22) and PMB2948(B24).

Therefore, both coprimary objectives were achieved at the 1.5 foldnoninferiority margin for all antigens in Tdap, MCV4, and bivalentrLP2086 vaccines.

Description of MCV4/Tdap Seroresponse Rates.

One of the secondary objectives of this study was to describeseroresponses to antigens contained in MCV4 and Tdap (Groups 1 and 2)when measured 1 month after vaccination with MCV4 and Tdap (Visit 2).

The seroresponse rates for the 10 Tdap and MVC4 antigens with thecorresponding 95% CIs in each group, and the percent differences (Group1-Group 2), and the 95% CIs of the differences are presented in Table Dfor the post-Vaccination 1 evaluable immunogenicity population.

For the evaluable immunogenicity population, the seroresponse rates forthe 6 Tdap antigens at 1 month after Vaccination 1 for Group 1 and Group2 were as follows: 98.6% and 98.3%, respectively (diphtheria toxoid);97.7% and 97.4%, respectively (tetanus toxoid); 68.1 and 72.7%,respectively (pertussis toxoid); 85.3% and 89.2%, respectively(pertussis filamentous hemagglutinin); 96.0% and 96.2%, respectively(pertussis pertactin); and 79.5% and 81.9%, respectively (pertussisfimbrial agglutinogens types 2+3). For the evaluable immunogenicitypopulation, the seroresponse rates for the 4 MCV4 antigens at 1 monthafter Vaccination 1 for Group 1 and Group 2 were as follows: 85.4% and88.6%, respectively (serogroup A); 89.3% and 88.9%, respectively(serogroup C); 90.5% and 93.6%, respectively (serogroup Y); and 97.1%and 97.2%, respectively (serogroup W-135). The difference inseroresponse rates of Group 1 to Group 2 at 1 month after Vaccination 1were 0.2% for diphtheria toxoid (95% CI: (−1.1, 1.6), 0.2% for tetanustoxoid (95% CI: −1.4, 1.8), −4.6% for pertussis toxoid (95% CI: −9.1,−0.1), −4.0% for pertussis filamentous hemagglutinin (95% CI: −7.3,−0.6), −0.2% for pertussis pertactin (95% CI: −2.1, 1.8), −2.5% forpertussis fimbrial agglutinogens types 2+3 (95% CI: −6.4, 1.5), −3.2%for serogroup A (95% CI: −6.7, 0.3), 0.3% for serogroup C (95% CI: −2.8,3.5), −3.2% for serogroup Y (95% CI: −6.0, −0.4), and −0.1% forserogroup W-135 (95% CI: −1.9, 1.7).

For the MCV4+Tdap+bivalent rLP2086 group (Group 1) and theMCV4+Tdap+saline group (Group 2), the estimated seroresponse ratedifferences were between −4.6% and 0.3% for the 6 Tdap and 4 MCV4antigens, with the lower bounds of the 95% CI of the rate difference−9.1% or greater (ie, all greater than −10%).

TABLE D Comparisons of Seroresponse Rates for Tdap/MCV4 Antigens at 1Month After Vaccination 1 - Post- Vaccination 1 Evaluable ImmunogenicityPopulation Vaccine Group (as Randomized) Group 1 Group 2 Tdap/MCV4MCV4 + Tdap + rLP2086 MCV4 + Tdap + Saline Difference Antigen N^(a)n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c) (%)^(d) (95% CI)^(e)Tdap Diphtheria 774 763 (98.6) (97.5, 99.3) 780 767 (98.3) (97.2, 99.1)0.2 (−1.1, 1.6)   Tetanus 774 756 (97.7) (96.3, 98.6) 780 760 (97.4)(96.1, 98.4) 0.2 (−1.4, 1.8)   Pertussis toxoid 774 527 (68.1) (64.7,71.4) 780 567 (72.7) (69.4, 75.8) −4.6 (−9.1, −0.1) Pertussisfilamentous 774 660 (85.3) (82.6, 87.7) 780 696 (89.2) (86.8, 91.3) −4.0(−7.3, −0.6) hemagglutinin Pertussis pertactin 774 743 (96.0) (94.4,97.3) 780 750 (96.2) (94.6, 97.4) −0.2 (−2.1, 1.8)   Pertussis fimbriae774 615 (79.5) (76.4, 82.3) 780 639 (81.9) (79.0, 84.6) −2.5 (−6.4,1.5)   agglutinogens types 2 + 3 rSBA MCV4 Serogroup A 712 608 (85.4)(82.6, 87.9) 736 652 (88.6) (86.1, 90.8) −3.2 (−6.7, 0.3)   Serogroup C738 659 (89.3) (86.8, 91.4) 742 660 (88.9) (86.5, 91.1) 0.3 (−2.8,3.5)   Serogroup Y 754 682 (90.5) (88.1, 92.5) 753 705 (93.6) (91.6,95.3) −3.2 (−6.0, −0.4) Serogroup W-135 729 708 (97.1) (95.6, 98.2) 752731 (97.2) (95.8, 98.3) −0.1 (−1.9, 1.7)   Abbreviations: ELISA =enzyme-linked immunosorbent assay; EU = ELISA units; LLOQ = lower limitof quantitation; MCV4 = quadrivalent meningococcal polysaccharideconjugate (vaccine); rSBA = serum bactericidal assay using rabbitcomplement; Tdap = tetanus, diphtheria, and acellular pertussis(vaccine). Note: Seroresponse is defined as: (1) For Tdap antigens,≥4-fold rise in antibody concentration if the prevaccination antibodyconcentration is ≤ the cutoff value, and ≥2-fold rise in antibodyconcentration if the prevaccination antibody concentration is > thecutoff value. (2) For MCV4, seroresponse is defined as ≥4-fold rise onrSBA titers if baseline value is ≥ LLOQ, and postdose rSBA titers ≥2 ×LLOQ if baseline value is < LLOQ. Note: Cutoff value = 0.1 IU/mL fordiphtheria; 0.1 IU/mL for tetanus; 0.9 EU/mL for pertussis toxoid; 2.9EU/mL for pertussis filamentous hemagglutinin; 3.0 EU/mL for pertussispertactin; 10.6 EU/mL for pertussis fimbriae agglutinogens types 2 + 3.LLOQ = 1:32 for MCV4 serogroup A, 1:64 for serogroup C, 1:128 forserogroup Y, and 1:64 for serogroup W-135. ^(a)N = number of subjectswith valid and determinate assay results for the given antigen at boththe specified time point and baseline. ^(b)n = Number of subjects withseroresponse. ^(c)Exact 2-sided confidence intervals (CIs) based uponobserved proportion of subjects using the Clopper and Pearson method.^(d)Difference in proportions, expressed as a percentage. ^(e)Exact2-sided CI (based on Chan and Zhang) for the difference in proportions,expressed as a percentage.Description of hSBA Titer 4-Fold Rise.

MCV4+Tdap+bivalent rLP2086 (Group 1) was compared tosaline+saline+bivalent rLP2086 (Group 3), by analyzing the hSBA titer4-fold response rates for 2 primary MnB strains (PMB80 [A22] and PMB2948[B24]) at 1 month after Vaccination 3. The number and proportion ofsubjects achieving an hSBA titer fold rise ≥4 from baseline to 1 monthafter Vaccination 3 for the 2 primary MnB strains with the corresponding95% CIs in each group, the percent differences (Group 1-Group 3) in theproportion, and the 95% CIs of the differences are presented in Table Efor the post-Vaccination 3 evaluable immunogenicity population.

The proportions of subjects achieving a ≥4-fold rise in hSBA titer frombaseline to 1 month after Vaccination 3 for the 2 primary MnB teststrains were measured for both Group 1 subjects who receivedMCV4+Tdap+bivalent rLP2086 and Group 3 subjects who receivedsaline+saline+bivalent rLP2086. Of the subjects in Group 1, 84.0%exhibited a ≥4-fold rise in hSBA titer against test strain PMB80 (A22),and 85.7% exhibited a ≥4-fold rise in hSBA titer against test strainPMB2948 (B24). Of the subjects in Group 3, 88.7% exhibited a ≥4-foldrise in hSBA titer against test strain PMB80 (A22) and 87.7% exhibited a≥4-fold rise in hSBA titer against test strain PMB2948 (B24).

The difference in the proportion of subjects achieving a ≥4-fold rise inhSBA titer between Group 1 and Group 3 at 1 month after Vaccination 3was −4.7% for PMB80 (A22) (95% CI: −8.4, −1.1) and −2.1% for PMB2948(B24) (95% CI: −5.7, 1.6). The estimated differences of 4-fold responserate were small (<5.0%), with the lower bounds of the 95% CI of theproportion difference being −8.4% (PMB80 [A22]) and −5.7% (PMB2948[B24]).

TABLE E Comparison of Subjects Achieving hSBA Titer Fold Rise ≥4 FromBaseline to 1 Month After Vaccination 3 - Post-Vaccination 3 EvaluableImmunogenicity Population Vaccine Group (as Randomized) Group 1 Group 3MCV4 + Tdap + rLP2086 Saline + Saline + rLP2086 Difference Strain(Variant) N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c)(%)^(d) (95% CI)^(e) PMB80 (A22) 673 565 (84.0) (81.0, 86.6) 672 596(88.7) (86.0, 91.0) −4.7 (−8.4, −1.1) PMB2948 (B24) 664 569 (85.7)(82.8, 88.3) 653 573 (87.7) (85.0, 90.2) −2.1 (−5.7, 1.6)  Abbreviations: hSBA = serum bactericidal assay using human complement;LLOQ = lower limit of quantitation; LOD = limit of detection. Note:Baseline is defined as the blood draw prior to Vaccination 1. Note: The4-fold increase is defined as follows: (1) For subjects with a baselinehSBA titer < LOD, or an hSBA titer < LLOQ, a 4-fold response is definedas an hSBA titer ≥4 times the LLOQ. (2) For subjects with a baselinehSBA titer ≥ LLOQ, a 4-fold response is defined as an hSBA titer ≥4times the baseline titer. ^(a)N = number of subjects with valid anddeterminate hSBA titers at baseline and at 1 month after Vaccination 3for the given strain. ^(b)n = Number of subjects achieving at least a4-fold response at 1 month after Vaccination 3 for the given strain.^(c)Exact 2-sided confidence interval (CI) based upon the observedproportion of subjects using the Clopper and Pearson method.^(d)Difference in proportions, expressed as a percentage. ^(e)Exact2-sided CI and corresponding p-value (based on Chan & Zhang) for thedifference in proportions, expressed as a percentage.Summary of Immune Response to Bivalent rLP2086 and MCV4 and TdapAntigens when Administered Concomitantly.

The noninferiority criteria for bivalent rLP2086 given with MCV4 andTdap vaccines compared to both MCV4 and Tdap vaccines alone or comparedto saline+saline+bivalent rLP2086 required that the lower limits of the2-sided 95% CI for the GMRs be greater than 0.67 for each of the 10MCV4/Tdap antigens (1 month after Vaccination 1) and for each of the 2primary MnB test strains (A22 and B24) (1 month after Vaccination 3).This prespecified threshold was met for both MnB test strains and eachof the 10 Tdap and MCV4 antigens.

For the MCV4+Tdap+bivalent rLP2086 group (Group 1) and theMCV4+Tdap+saline group (Group 2), the estimated seroresponse ratedifferences were between −4.6% and 0.3% for the 6 Tdap and 4 MCV4antigens, with the lower bounds of the 95% CI of the rate differenceequal to 9.1% or less (all differences were greater than −10%). The≥4-fold rise hSBA responses to 2 primary MnB test strains (A22 and B24)were similar (ranging from 84.0% to 88.7%) between the group thatreceived MCV4+Tdap+bivalent rLP2086 and the group that receivedsaline+saline+bivalent rLP2086.

Immune Response to Bivalent rLP2086. hSBA Titers LLOQ (DescriptiveEndpoint).

One of the secondary objectives of this study was to describe the immuneresponse as measured by hSBA performed with 2 primary MnB test strains,1 expressing a subfamily A LP2086 (PMB80 [A22]) and 1 expressing asubfamily B LP2086 (PMB2948 [B24]), measured 1 month after the second(Visit 4) and the third (Visit 6) vaccinations with bivalent rLP2086.

One of the descriptive secondary endpoints for this secondary objectivewas the proportion of subjects with hSBA titers ≥LLOQ at 1 month afterVaccination 2 (Visit 4) and at 1 month after Vaccination 3 (Visit 6) toeach of the 2 primary MnB test strains. The proportion of subjects withan hSBA titer ≥LLOQ to each of the 2 primary MnB test strains for thepost-Vaccination 3 evaluable immunogenicity population is presented inTable F. Note that the LLOQ for PMB80 (A22) was an hSBA titer equal to1:16, while the LLOQ for PMB2948 (B24) was an hSBA titer equal to 1:8.

For Group 3 (saline+saline+bivalent rLP2086), the proportion of subjectswith an hSBA titer ≥LLOQ at baseline (before Vaccination 1) was 5.6% forPMB80 (A22) and 3.4% for PMB2948 (B24). For Group 3(saline+saline+bivalent rLP2086), the proportions of subjects achievingan hSBA titer ≥LLOQ at 1 month after Vaccination 2 and at 1 month afterVaccination 3 were 68.0% and 91.4%, respectively, for PMB80 (A22) and66.0% and 92.7%, respectively, for PMB2948 (B24).

For Group 1 (MCV4+Tdap+bivalent rLP2086), the proportion of subjectswith an hSBA titer ≥LLOQ at baseline (before Vaccination 1) was 4.4% forPMB80 (A22) and 1.6% for PMB2948 (B24). For Group 1 (MCV4+Tdap+bivalentrLP2086), the proportions of subjects achieving an hSBA titer ≥LLOQ at 1month after Vaccination 2 and at 1 month after Vaccination 3 were 68.0%and 87.5%, respectively, for PMB80 (A22); and 62.3% and 90.0%,respectively, for PMB2948 (B24).

Compared to before Vaccination 1, a substantial increase in theproportion of subjects with an hSBA titer ≥LLOQ for the 2 primary MnBtest strains was observed among both Group 1 and Group 3 subjects at 1month after Vaccination 2, with additional increases observed at 1 monthafter Vaccination 3. The hSBA responses in Groups 1 and 3 of theevaluable immunogenicity population were similar.

TABLE F Subjects With hSBA Titer ≥ LLOQ - Post-Vaccination 3 EvaluableImmunogenicity Population Vaccine Group (as Randomized) Group 1 Group 3Strain (Variant) MCV4 + Tdap + rLP2086 Saline + Saline + rLP2086 TotalSampling Time Point N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95%CI)^(c) N^(a) n^(b) (%) (95% CI)^(c) PMB80 (A22) Before Vaccination 1677  30 (4.4)  (3.0, 6.3) 677  38 (5.6)  (4.0, 7.6) 1354  68 (5.0) (3.9, 6.3) 1 Month after Vaccination 2 669 455 (68.0) (64.3, 71.5) 665452 (68.0) (64.3, 71.5) 1334  907 (68.0) (65.4, 70.5) 1 Month afterVaccination 3 679 594 (87.5) (84.8, 89.9) 674 616 (91.4) (89.0, 93.4)1353 1210 (89.4) (87.7, 91.0) PMB2948 (B24) Before Vaccination 1 677  11(1.6)  (0.8, 2.9) 676  23 (3.4)  (2.2, 5.1) 1353  34 (2.5)  (1.7, 3.5) 1Month after Vaccination 2 656 409 (62.3) (58.5, 66.1) 650 429 (66.0)(62.2, 69.6) 1306  838 (64.2) (61.5, 66.8) 1 Month after Vaccination 3670 603 (90.0) (87.5, 92.2) 656 608 (92.7) (90.4, 94.6) 1326 1211 (91.3)(89.7, 92.8) Abbreviations: hSBA = serum bactericidal assay using humancomplement; LLOQ = lower limit of quantitation. Note: LLOQ = 1:16 forA22; 1:8 for B24. ^(a)N = number of subjects with valid and determinatehSBA titers for the given strain. ^(b)n = Number of subjects withobserved hSBA titer ≥ LLOQ for the given strain at the given time point.^(c)Exact 2-sided confidence interval (CI) based upon the observedproportion of subjects using the Clopper and Pearson method.hSBA Titers Achieving a Defined Level

Another secondary endpoint was the proportion of subjects with hSBAtiters LLOQ, ≥1:4, ≥1:8, ≥1:16, ≥1:32, ≥1:64, and ≥1:128 to each of the2 primary MnB test strains at each blood sampling time point.

Subjects who achieved an hSBA titer ≥1:4 and ≥1:16 are described below.An hSBA titer of ≥1:4 is widely recognized as the correlate ofprotection against IMD; however, a more conservative hSBA titer of ≥1:16has been been considered a level indicative of a 4-fold vaccine effectfor subjects seronegative at prevaccination.

The proportions of subjects in Group 1 (MCV4+Tdap+bivalent rLP2086) andGroup 3 (saline+saline+bivalent rLP2086) with prevaccination (ie, beforeVaccination 1) hSBA titers of ≥1:4 were 6.9% and 7.5%, respectively, forstrain PMB80 (A22); and 2.1% and 3.7%, respectively, for strain PMB2948(B24). In addition, the proportions of subjects in Groups 1 and 3 withprevaccination hSBA titers of ≥1:16 were 4.4% and 5.6%, respectively,for strain PMB80 (A22); and 1.5% and 2.1%, respectively, for strainPMB2948 (B24).

In Group 3 (saline+saline+bivalent rLP2086), the proportion of subjectswith an hSBA titer ≥1:4 at 1 month after Vaccination 2 was 68.6% forPMB80 (A22) and 68.2% for PMB2948 (B24). One (1) month after Vaccination3, the proportion of subjects with an hSBA titer of ≥1:4 was 91.7% forPMB80 (A22) and 93.1% for PMB2948 (B24). In Group 1 (MCV4+Tdap+bivalentrLP2086), the proportion of subjects with an hSBA titer of ≥1:4 at 1month after Vaccination 2 was 68.2% for PMB80 (A22) and 64.8% forPMB2948 (B24). One (1) month after Vaccination 3, the proportion ofsubjects with an hSBA titer of ≥1:4 was 87.8% for PMB80 (A22) and 90.7%for PMB2948 (B24).

In Group 3 (saline+saline+bivalent rLP2086), the proportion of subjectswith an hSBA titer of ≥1:16 at 1 month after Vaccination 2 was 68.0% forPMB80 (A22) and 60.3% for PMB2948 (B24). One (1) month after Vaccination3, the proportion of subjects with an hSBA titer of ≥1:16 was 91.4% forPMB80 (A22) and 88.9% for PMB2948 (B24). In Group 1 (MCV4+Tdap+bivalentrLP2086), the proportion of subjects with an hSBA titer of ≥1:16 at 1month after Vaccination 2 was 68.0% for PMB80 (A22) and 57.6% forPMB2948 (B24). One (1) month after Vaccination 3, the proportion ofsubjects with an hSBA titer of ≥1:16 was 87.5% for PMB80 (A22) and 86.7%for PMB2948 (B24).

For both Group 1 and Group 3, the majority of subjects achieved an hSBAtiter of 1:16 or greater following 2 or 3 doses of bivalent rLP2086,while relatively few subjects had a measurable hSBA titer to any of theprimary MnB test strains at prevaccination Visit 1.

hSBA Geometric Mean Titers.

Another secondary endpoint was the hSBA GMTs for each of the 2 primaryMnB test strains at each applicable blood sampling time point. Table Gpresents the hSBA GMTs for each of the 2 primary MnB test strains andthe corresponding CIs by sampling time point for Group 1 and Group 3 inthe post-Vaccination 3 evaluable immunogenicity population. The GMTs atbaseline were below the hSBA LLOQs for both groups. For Group 3(saline+saline+bivalent rLP2086), hSBA GMTs at 1 month after Vaccination2 were 23.8 for PMB80 (A22) and 13.0 for PMB2948 (B24). The hSBA GMTs at1 month after Vaccination 3 were 49.7 for PMB80 (A22) and 27.4 forPMB2948 (B24). For Group 1 (MCV4+Tdap+bivalent rLP2086), hSBA GMTs at 1month after Vaccination 2 were 23.7 for PMB80 (A22) and 12.0 for PMB2948(B24). The hSBA GMTs at 1 month after Vaccination 3 were 45.9 for PMB80(A22) and 24.8 for PMB2948 (B24). The hSBA responses as measured by GMTwere similar between Groups 1 and 3.

Reverse Cumulative Distribution Curves for hSBA Titers

The Reverse Cumulative Distribution Curves (RCDCs) showing thedistribution of hSBA titers for PMB80 (A22) and PMB2948 (B24) wereassessed for Groups 1 and 3 at all sampling time points for thepost-Vaccination 3 evaluable immunogenicity population. The RCDCs showedthat the majority of subjects responded after Vaccination 2 and had anadditional increase in titer for the 2 primary MnB test strains afterVaccination 3. Immune responses to the antigens were similar for Groups1 and 3.

hSBA≥4-Fold Response

A prespecified, exploratory objective of this study was to describe theimmune response to bivalent rLP2086, as measured by a 4-fold response inhSBA titer performed with 2 primary MnB test strains, 1 expressingLP2086 subfamily A proteins and 1 expressing LP2086 subfamily Bproteins, measured at 1 month after the second and third vaccinations(Visit 3 and Visit 5) with bivalent rLP2086.

The endpoints for the exploratory objective were the proportion ofsubjects achieving at least a 4-fold increase in hSBA titer to 2 MnBstrains, PMB80 (A22) and PMB2948 (B24), from baseline to 1 month afterthe second and third vaccinations with bivalent rLP2086. Note that theLLOQ for PMB80 (A22) was an hSBA titer equal to 1:16, while the LLOQ forPMB80 (A22) was an hSBA titer equal to 1:8. The 4-fold response wasdefined as follows: For subjects with a baseline hSBA titer below thelimit of detection (LOD) or an hSBA titer of <1:4, a 4-fold response wasdefined as an hSBA titer of ≥1:16. For subjects with a baseline hSBAtiter of ≥LOD (ie, hSBA titer of ≥1:4) and <LLOQ, a 4-fold response wasdefined as an hSBA titer ≥4 times the LLOQ. For subjects with a baselinehSBA titer of ≥LLOQ, a 4-fold response was defined as an hSBA titer of≥4 times the baseline titer.

The proportion of subjects achieving an hSBA titer fold rise ≥4 for eachof the 2 primary MnB test strains for the post-Vaccination 3 evaluableimmunogenicity population are presented for Group 1 (MCV4+Tdap+bivalentrLP2086) and Group 3 (saline+saline+bivalent rLP2086) in Table H.

For Group 3 (saline+saline+bivalent rLP2086), the proportion of subjectsachieving an hSBA titer fold rise ≥4 from baseline to 1 month afterVaccination 3 was 88.7% for PMB80 (A22) and 87.7% for PMB2948 (B24). At1 month after Vaccination 2, the proportion of subjects achieving anhSBA titer fold rise ≥4 from baseline was 63.7% for PMB80 (A22) and58.4% for PMB2948 (B24).

For Group 1 (MCV4+Tdap+bivalent rLP2086), the proportion of subjectsachieving an hSBA titer fold rise ≥4 from baseline to 1 month afterVaccination 3 was 84.0% for PMB80 (A22) and 85.7% for PMB2948 (B24). At1 month after Vaccination 2, the proportion of subjects achieving anhSBA titer fold rise ≥4 from baseline was 64.3% for PMB80 (A22) and56.3% for PMB2948 (B24).

The hSBA responses as measured by 4-fold titer response were similarbetween Groups 1 and 3.

TABLE G hSBA Geometric Mean Titers - Post-Vaccination 3 EvaluableImmunogenicity Population Vaccine Group (as Randomized) Group 1 Group 3Strain (Variant) MCV4 + Tdap + rLP2086 Saline + Saline + rLP2086 TotalSampling Time Point N^(a) GMT^(b) (95% CI)^(c) N^(a) GMT^(b) (95%CI)^(c) N^(a) GMT^(b) (95% CI)^(c) PMB80 (A22) Before Vaccination 1 677 8.5 (8.28, 8.64) 677  8.6 (8.39, 8.84) 1354  8.5 (8.39, 8.68) 1 Monthafter Vaccination 2 669 23.7 (22.10, 25.40) 665 23.8 (22.14, 25.54) 133423.7 (22.58, 24.95) 1 Month after Vaccination 3 679 45.9 (42.74, 49.35)674 49.7 (46.43, 53.30) 1353 47.8 (45.47, 50.23) PMB2948 (B24) BeforeVaccination 1 677  4.1 (4.04, 4.19) 676  4.2 (4.10, 4.27) 1353  4.2(4.09, 4.21) 1 Month after Vaccination 2 656 12.0 (11.12, 12.99) 65013.0 (12.03, 14.13) 1306 12.5 (11.83, 13.23) 1 Month after Vaccination 3670 24.8 (23.11, 26.58) 656 27.4 (25.58, 29.41) 1326 26.1 (24.80, 27.38)Abbreviations: GMT = geometric mean titer; hSBA = serum bactericidalassay using human complement; LLOQ = lower limit of quantitation. Note:LLOQ = 1:16 for A22; 1:8 for B24. Titers below the LLOQ were set to 0.5× LLOQ for analysis. ^(a)N = number of subjects with valid anddeterminate hSBA titers for the given strain. ^(b)GMTs were calculatedusing all subjects with valid and determinate hSBA titers at the giventime point. ^(c)Confidence intervals (CIs) are back transformations ofconfidence levels based on the Student t distribution for the meanlogarithm of assay results.

TABLE H Subjects Achieving ≥4-Fold Rise in hSBA Titer - Post-Vaccination3 Evaluable Immunogenicity Population Vaccine Group (as Randomized)Group 1 Group 3 Strain (Variant) MCV4 + Tdap + rLP2086 Saline + Saline +rLP2086 Total Sampling Time Point N^(a) n^(b) (%) (95% CI)^(c) N^(a)n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c) hSBA Titer fold rise≥4 from baseline PMB80 (A22) 1 Month after Vaccination 2 663 426 (64.3)(60.5, 67.9) 663 422 (63.7) (59.9, 67.3) 1326  848 (64.0) (61.3, 66.5) 1Month after Vaccination 3 673 565 (84.0) (81.0, 86.6) 672 596 (88.7)(86.0, 91.0) 1345 1161 (86.3) (84.4, 88.1) PMB2948 (B24) 1 Month afterVaccination 2 650 366 (56.3) (52.4, 60.2) 647 378 (58.4) (54.5, 62.3)1297  744 (57.4) (54.6, 60.1) 1 Month after Vaccination 3 664 569 (85.7)(82.8, 88.3) 653 573 (87.7) (85.0, 90.2) 1317 1142 (86.7) (84.8, 88.5)Abbreviations: hSBA = serum bactericidal assay using human complement;LLOQ = lower limit of quantitation; LOD = limit of detection. Note: LLOQ= 1:16 for A22; 1:8 for B44. Note: Baseline is defined as the blood drawprior to Vaccination 1. Note: The 4-fold increase is defined as follows:(1) For subjects with a baseline hSBA titer < LOD (hSBA titer <1:4), aresponse is defined as an hSBA titer ≥1:16. (2) For subjects with abaseline hSBA titer ≥ LOD (hSBA titer ≥1:4) and < LLOQ, a response isdefined as an hSBA titer ≥4 times the LLOQ. (3) For subjects with abaseline hSBA titer ≥ LLOQ, a 4-fold response is defined as an hSBAtiter ≥4 times the baseline titer. ^(a)N = number of subjects with validand determinate hSBA titers for the given strain at both the specifiedtime point and baseline. ^(b)n = number of subjects who achieved hSBAtiter fold rise ≥4 from baseline for the given strain. ^(c)Exact 2-sidedconfidence interval (CI) based upon the observed proportion of subjectsusing the Clopper and Pearson method.Additional hSBA Fold Response

An additional endpoint was the proportion of subjects achieving at leasta 2-fold increase and the proportion of subjects achieving at least a3-fold increase in hSBA titer from baseline at each postvaccinationblood sampling visit for each of the 2 primary MnB strains. Note thatthe LLOQ for PMB80 (A22) was an hSBA titer equal to 1:16, while the LLOQfor PMB2948 (B24) was an hSBA titer equal to 1:8.

The proportions of subjects achieving a 2-fold rise in hSBA titer frombaseline to 1 month after Vaccination 2 for Group 1 and Group 3 for MnBstrains were 66.2% and 66.1%, respectively, for PMB80 (A22); and 56.8%and 59.7%, respectively, for PMB2948 (B24). The proportions of subjectsachieving an hSBA titer fold rise from baseline to 1 month afterVaccination 3 for Group 1 and Group 3 for MnB strains were 86.6% and90.3%, respectively, for PMB80 (A22); and 86.1% and 88.5%, respectively,for PMB2948 (B24).

The proportions of subjects achieving a ≥3 fold rise in hSBA titer frombaseline to 1 month after Vaccination 2 for Group 1 and Group 3 for MnBstrains were 64.3% and 63.7%, respectively, for PMB80 (A22); and 56.3%and 58.4%, respectively, for PMB2948 (B24). The proportions of subjectsachieving a ≥3 fold rise in hSBA titer from baseline to 1 month afterVaccination 3 for Group 1 and Group ≥3 for MnB strains were 84.0% and88.7%, respectively, for PMB80 (A22); and 85.7% and 87.7%, respectively,for PMB2948 (B24).

For subjects with baseline titer below LOD (1:4) in the post-Vaccination3 evaluable immunogenicity population, the majority of subjects achieveda 2-fold, 3-fold, and 4-fold response in both Group 1 and Group 3 at allblood sampling time points, ranging from 56.7% to 66.6% 1 month afterVaccination 2, and from 86.3% to 91.0% 1 month after Vaccination 3.

Summary of Immune Response to Bivalent rLP2086

In summary of the descriptive endpoints under the secondary objectives,the majority of subjects achieved an hSBA titer ≥LLOQ for both Group 1(MCV4+Tdap+bivalent rLP2086) and Group 3 (saline+saline+bivalentrLP2086) to both primary MnB test strains, while only a very smallproportion of subjects had measurable hSBA titers LLOQ at baseline(prevaccination Visit 1). Substantial immune responses with the 2 MnBstrains were observed at 1 month after Vaccination 2, with additionalincreases observed at 1 month after Vaccination 3 for both Group 1 andGroup 3 subjects. This conclusion was confirmed by the proportion ofsubjects with an hSBA titer of ≥1:16 following 3 doses, the observedGMTs achieved after 2 doses and after 3 doses in both groups, and theRCDCs for the 2 primary MnB test strains.

For both Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 3(saline+saline+bivalent rLP2086), a high proportion of subjects achievedan hSBA titer fold rise ≥4 for each of the 2 primary MnB test strains.

In addition, the majorities of subjects achieved an hSBA titer fold rise≥2 and an hSBA titer fold rise ≥3 for the 2 primary MnB strains at allsampling time points for both Group 1 (MCV4+Tdap+bivalent rLP2086) andGroup 3 (saline+saline+bivalent rLP2086). The proportion of subjectswith results meeting these criteria was higher after 3 vaccinationscompared to 2 vaccinations. Sensitivity analyses also support the robustimmune responses induced by 3 doses for both groups.

These results support the evidence that the immune response to bivalentrLP2086 when coadministered with the MCV4+Tdap vaccines yields a robustimmune response that is comparable to the immune response to bivalentrLP2086+saline.

Immune Response to MCV4 and Tdap Tdap/MCV4 Antigen GMTs and GMCs

Table I presents the GMTs and GMCs and the corresponding CIs for each ofthe Tdap and MCV4 antigens at baseline and 1 month after Vaccination 1for Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 2 (MCV4+Tdap+saline)in the post-Vaccination 1 evaluable immunogenicity population.

For Group 2 (MCV4+Tdap+saline), the Tdap antigen GMCs at baseline(before Vaccination 1) and at 1 month after Vaccination 1 were 0.2 and9.8, respectively (diphtheria toxoid); 0.5 and 10.3, respectively(tetanus toxoid); 3.9 and 14.2, respectively (pertussis toxoid); 21.8and 122.9, respectively (pertussis filamentous hemagglutinin); 16.2 and228.9, respectively (pertussis pertactin); 8.5 and 154.2, respectively(pertussis fimbrial agglutinogens types 2+3); and the MCV4 antigen GMTsat baseline (before Vaccination 1) and at 1 month after Vaccination 1were 436.3 and 5113.0, respectively (serogroup A); 84.3 and 1650.2,respectively (serogroup C); 168.5 and 2244.9, respectively (serogroupY); and 177.7 and 6367.9, respectively (serogroup W-135). For Group 1(MCV4+Tdap+bivalent rLP2086), the Tdap antigen GMs at baseline (beforeVaccination 1) and at 1 month after Vaccination 1 were 0.2 and 9.3,respectively (diphtheria toxoid); 0.5 and 9.4, respectively (tetanustoxoid); 3.9 and 13.2, respectively (pertussis toxoid); 21.3 and 112.0,respectively (pertussis filamentous hemagglutinin); 15.7 and 202.0,respectively (pertussis pertactin); 8.4 and 138.1, respectively(pertussis fimbrial agglutinogens types 2+3); and the MCV4 antigen GMsat baseline (before Vaccination 1) and at 1 month after Vaccination 1were 490.0 and 4647.3, respectively (serogroup A); 87.6 and 1679.2,respectively (serogroup C); 183.3 and 2212.6, respectively (serogroupY); and 176.7 and 5925.1, respectively (serogroup W-135). Overall, theestimated GMs were similar between Group 1 and Group 2.

IgG MCV4 GMCs

An exploratory objective of this study was to describe the IgG immuneresponse as measured by anti-MCV4 IgG GMCs induced by the MCV4 vaccinewhen given with and without bivalent rLP2086 as measured by anti-MCV IgGGMCs. Exploratory immunogenicity endpoints for this study were appliedto the 4 MCV4 antigenic components in subjects from Group 1(MCV4+Tdap+bivalent rLP2086) and Group 2 (MCV4+Tdap+saline). Theexploratory endpoints were the IgG response (measured as GMCs) toserogroup A, C, Y or W-135 plysaccharide. Following the validation ofthis assay (ie, postvalidation) and prior to sample testing, the LLOQswere adjusted upwards based on additional data analyses from standardreference curves to: 0.1795 μg/mL for serogroup A, 0.0940 μg/mL forserogroup C, 0.2485 μg/mL for serogroup Y, and 0.2530 μg/mL forserogroup W-135. Results below the LLOQ were set to 0.5×LLOQ foranalysis. MCV4 IgG GMCs for Group 1 and Group 2 in the post-Vaccination1 evaluable immunogenicity population using the adjusted postvalidationLLOQs are presented in Table J.

For Group 1 (MCV4+Tdap+rLP2086), the MCV4 IgG GMCs at baseline (beforeVaccination 1) and at 1 month after Vaccination 1 were: 0.17 and 11.42μg/mL, respectively (serogroup A); 0.11 and 5.59 μg/mL, respectively(serogroup C); 0.14 and 2.49 μg/mL, respectively (serogroup Y); and 0.13and 1.79 μg/mL, respectively (serogroup W-135). For Group 2(MCV4+Tdap+saline), the MCV4 IgG GMCs at baseline (before Vaccination 1)and at 1 month after Vaccination 1 were: 0.15 and 11.38 μg/mL,respectively (serogroup A); 0.11 and 5.47 μg/mL, respectively (serogroupC); 0.13 and 2.14 μg/mL, respectively (serogroup Y); and 0.13 and 1.84μg/mL, respectively (serogroup W-135).

Overall, the MCV4 IgG GMCs were numerically similar for Group 1 andGroup 2. Based on the initial assay validation data analyses, the MCV4IgG assay LLOQs were set at 0.1037 μg/mL for serogroup A, 0.0204 μg/mLfor serogroup C, 0.1104 μg/mL for serogroup Y, and 0.1198 μg/mL forserogroup W-135. MCV4 IgG GMCs for Group 1 and Group 2 in thepost-Vaccination 1 evaluable immunogenicity population using thevalidation LLOQs also showed that the MCV4 IgG GMCS were numericallysimilar for Group 1 and Group 2.

Subjects Achieving Prespecified Antibody Level for DiphtheriaToxoid/Tetanus Antigens

A descriptive secondary endpoint of the study was the proportion ofsubjects (Groups 1 and 2) who achieve an antibody level ≥1.0 IU/mL totetanus toxoid and proportion of subjects (Groups 1 and 2) who achievean antibody level ≥1.0 IU/mL to diphtheria toxoid.

The proportion of subjects achieving the prespecified level ofantibodies to diphtheria toxoid and tetanus toxoid antigens, withcorresponding 95% CI are presented in Table K for Group 1(MCV4+Tdap+bivalent rLP2086) and Group 2 (MCV4+Tdap+saline) in thepost-Vaccination 1 evaluable immunogenicity population.

For Group 1 (MCV4+Tdap+bivalent rLP2086), the proportion of subjectsachieving the prespecified level of antibodies to diphtheria toxoid andtetanus toxoid antigens 1 month after Vaccination 1 was 98.1% (95% CI:96.8, 98.9) and 99.1% (95% CI: 98.2, 99.6), respectively.

For Group 2 (MCV4+Tdap+saline), the proportion of subjects achieving theprespecified level of antibodies to diphtheria toxoid and tetanusantigens 1 month after Vaccination 1 was 99.0% (95% CI: 98.0, 99.6) and99.0% (95% CI: 98.0, 99.6), respectively.

TABLE I Tdap/MCV4 Antigen Geometric Means - Post-Vaccination 1 EvaluableImmunogenicity Population Vaccine Group (as Randomized) Group 1 Group 2Tdap/MCV4 Antigen MCV4 + Tdap + rLP2086 MCV4 + Tdap + Saline TotalSampling Time Point N^(a) GM^(b) (95% CI)^(c) N^(a) GM^(b) (95% CI)^(c)N^(a) GM^(b) (95% CI)^(c) Tdap Diphtheria Before Vaccination 1 775 0.2(0.15, 0.18) 781 0.2 (0.15, 0.17) 1556 0.2 (0.15, 0.17) 1 Month afterVaccination 1 778 9.3 (8.67, 9.92) 780 9.8  (9.23, 10.51) 1558 9.6 (9.12, 10.02) Tetanus Before Vaccination 1 775 0.5 (0.43, 0.50) 781 0.5(0.44, 0.52) 1556 0.5 (0.44, 0.50) 1 Month after Vaccination 1 778 9.4(8.95, 9.98) 780 10.3  (9.75, 10.85) 1558 9.9  (9.49, 10.24) Pertussistoxoid Before Vaccination 1 775 3.9 (3.48, 4.26) 781 3.9 (3.54, 4.29)1556 3.9 (3.62, 4.15) 1 Month after Vaccination 1 778 13.2 (12.35,14.14) 780 14.2 (13.28, 15.20) 1558 13.7 (13.06, 14.37) Pertussisfilamentous hemagglutinin Before Vaccination 1 775 21.3 (19.66, 23.12)781 21.8 (20.11, 23.62) 1556 21.6 (20.36, 22.82) 1 Month afterVaccination 1 778 112.0 (106.15, 118.14) 780 122.9 (116.42, 129.84) 1558117.3 (112.94, 121.92) Pertussis pertactin Before Vaccination 1 775 15.7(14.29, 17.30) 781 16.2 (14.81, 17.66) 1556 15.9 (14.95, 17.02) 1 Monthafter Vaccination 1 778 202.0 (187.77, 217.25) 780 228.9 (212.72,246.35) 1558 215.0 (204.20, 226.47) Pertussis fimbriae agglutinogenstypes 2 + 3 Before Vaccination 1 775 8.4 (7.88, 9.02) 781 8.5 (7.94,9.14) 1556 8.5 (8.07, 8.90) 1 Month after Vaccination 1 778 138.1(121.20, 157.33) 780 154.2 (135.30, 175.79) 1558 145.9 (133.07, 160.06)rSBA MCV4 Serogroup A Before Vaccination 1 726 490.0 (450.15, 533.38)745 436.3 (401.66, 473.89) 1471 462.0 (435.45, 490.20) 1 Month afterVaccination 1 763 4647.3 (4317.66, 5002.09) 772 5113.0 (4748.73,5505.17) 1535 4875.9 (4628.23, 5136.92) Serogroup C Before Vaccination 1748 87.6 (79.78, 96.23) 756 84.3 (77.00, 92.38) 1504 86.0 (80.53, 91.75)1 Month after Vaccination 1 768 1679.2 (1539.63, 1831.38) 767 1650.2(1519.01, 1792.65) 1535 1664.6 (1567.82, 1767.39) Serogroup Y BeforeVaccination 1 762 183.3 (170.25, 197.43) 764 168.5 (157.41, 180.37) 1526175.8 (167.13, 184.82) 1 Month after Vaccination 1 771 2212.6 (2056.08,2381.08) 770 2244.9 (2088.70, 2412.89) 1541 2228.7 (2117.07, 2346.26)Serogroup W-135 Before Vaccination 1 755 176.7 (161.93, 192.74) 767177.7 (163.47, 193.17) 1522 177.2 (166.83, 188.19) 1 Month afterVaccination 1 751 5925.1 (5469.77, 6418.33) 765 6367.9 (5872.68,6904.88) 1516 6144.6 (5804.87, 6504.11) Abbreviations: ELISA =enzyme-linked immunosorbent assay; EU = ELISA units; GM = geometricmean; LLOQ = lower limit of quantitation; MCV4 = quadrivalentmeningococcal polysaccharide conjugate (vaccine); rSBA = serumbactericidal assay using rabbit complement; Tdap = tetanus, diphtheria,and acellular pertussis (vaccine). Note: LLOQ = 0.037 IU/mL fordiphtheria; 0.05 IU/mL for tetanus; 0.9 EU/mL for pertussis toxoid; 2.9EU/mL for pertussis filamentous hemagglutinin; 3.0 EU/mL for pertussispertactin; 10.6 EU/mL for pertussis fimbriae agglutinogens types 2 + 3.1:32 for rSBA serogroup A, 1:64 for serogroup C, 1:128 for serogroup Y,and 1:64 for serogroup W-135. Results below the LLOQ were set to 0.5 ×LLOQ for analysis. ^(a)N = number of subjects with valid and determinateassay results for the given antigen. ^(b)GMs were calculated using allsubjects with valid and determinate assay results. ^(c)Confidenceintervals (CIs) are back transformations of confidence levels based onthe Student t distribution for the mean logarithm of assay results.

TABLE J IgG MCV4 Geometric Mean Concentrations (Postvalidation LLOQs) -Post-Vaccination 1 Evaluable Immunogenicity Population Vaccine Group (asRandomized) Group 1 Group 2 Antigen MCV4 + Tdap + rLP2086 MCV4 + Tdap +Saline Total Sampling Time Point N^(a) GMC^(b) (95% CI)^(c) N^(a)GMC^(b) (95% CI)^(c) N^(a) GMC^(b) (95% CI)^(c) Serogroup A BeforeVaccination 1 779  0.17 (0.16, 0.19) 781  0.15 (0.14, 0.17) 1560  0.16(0.15, 0.17) 1 Month after Vaccination 1 779 11.42 (10.30, 12.65) 78111.38 (10.21, 12.67) 1560 11.40 (10.58, 12.28) Serogroup C BeforeVaccination 1 779  0.11 (0.10, 0.12) 781  0.11 (0.10, 0.12) 1560  0.11(0.10, 0.12) 1 Month after Vaccination 1 779  5.59 (4.90, 6.39) 781 5.47 (4.79, 6.23) 1560  5.53 (5.04, 6.07) Serogroup Y BeforeVaccination 1 779  0.14 (0.13, 0.14) 781  0.13 (0.13, 0.14) 1560  0.14(0.13, 0.14) 1 Month after Vaccination 1 779  2.49 (2.22, 2.79) 781 2.14 (1.92, 2.39) 1560  2.31 (2.13, 2.50) Serogroup W-135 BeforeVaccination 1 779  0.13 (0.13, 0.14) 781  0.13 (0.13, 0.14) 1560  0.13(0.13, 0.14) 1 Month after Vaccination 1 779  1.79 (1.59, 2.01) 781 1.84 (1.62, 2.09) 1560  1.81 (1.66, 1.98) Abbreviations: GMC =geometric mean concentration; IgG = immunoglobulin G; LLOQ = lower limitof quantitation. Note: LLOQ = 0.1795 μg/mL for IgG MCV4 serogroup A,0.0940 μg/mL for IgG MCV4 serogroup C, 0.2485 μg/mL for IgG MCV4serogroup Y, and 0.2530 μg/mL for IgG MCV4 serogroup W-135. Resultsbelow the LLOQ were set to 0.5 × LLOQ for analysis. ^(a)N = number ofsubjects with valid and determinate assay results for the givenserogroup. ^(b)GMCs were calculated using all subjects with valid anddeterminate assay results. ^(c)Confidence intervals (CIs) are backtransformations of confidence levels based on the Student t distributionfor the mean logarithm of assay results.

TABLE K Subjects Achieving Prespecified Antibody Level for DiphtheriaToxoid/Tetanus Antigens - Post- Vaccination 1 Evaluable ImmunogenicityPopulation Vaccine Group (as Randomized) Group 1 Group 2 AntigenAntibody MCV4 + Tdap + rLP2086 MCV4 + Tdap + Saline Total Sampling TimePoint Level N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c)N^(a) n^(b) (%) (95% CI)^(c) Diphtheria toxoid Before Vaccination 1 ≥1.0IU/mL 775  32 (4.1)  (2.8, 5.8) 781 35 (4.5) (3.1, 6.2) 1556  67 (4.3)(3.4, 5.4) 1 Month after Vaccination 1 ≥1.0 IU/mL 778 763 (98.1) (96.8,98.9) 780 772 (99.0) (98.0, 99.6) 1558 1535 (98.5) (97.8, 99.1) TetanusBefore Vaccination 1 ≥1.0 IU/mL 775 202 (26.1) (23.0, 29.3) 781 205(26.2) (23.2, 29.5) 1556  407 (26.2) (24.0, 28.4) 1 Month afterVaccination 1 ≥1.0 IU/mL 778 771 (99.1) (98.2, 99.6) 780 772 (99.0)(98.0, 99.6) 1558 1543 (99.0) (98.4, 99.5) ^(a)N = number of subjectswith valid and determinate assay results for the given antigen. ^(b)n =Number of subjects with antibody concentration prespecified level forthe given antigen. ^(c)Exact 2-sided confidence intervals (CIs) basedupon the observed proportion of subjects using the Clopper and Pearsonmethod.

RCDCs for MCV4 and Tdap Antigens

The RCDCs showing the distribution of rSBA titers for MCV4 serogroups A,C, Y, and W-135 for Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 2(MCV4+Tdap+saline), before and 1 month after Vaccination 1, for thepost-Vaccination 1 evaluable immunogenicity population showed robustimmune responses among subjects after Vaccination 1 for both groups andthe curves were similar throughout the range of rSBA titers.

The RCDCs showing the distribution of IgG titers to MCV4 serogroup A, C,Y, and W-135 polysaccharides using the postvalidation LLOQs for Group 1(MCV4+Tdap+bivalent rLP2086) and Group 2 (MCV4+Tdap+saline), before and1 month after Vaccination 1, for the post-Vaccination 1 evaluableimmunogenicity population, and the RCDCs showing the distribution of IgGtiters for MCV4 serogroups A, C, Y, and W-135 using the validation LLOQsfor Group 1 (MCV4+Tdap+bivalent rLP2086) and Group 2 (MCV4+Tdap+saline),before and 1 month after Vaccination 1, for the post-Vaccination 1evaluable immunogenicity population showed robust immune responses amongsubjects after Vaccination 1 for both groups using both the validationand postvalidation LLOQs.

The RCDCs showing the distribution of Tdap titers for diphtheria,tetanus, pertussis toxoid, pertussis filamentous hemagglutinin,pertussis pertactin, and pertussis fimbrial agglutinogens types 2+3 forGroup 1 (MCV4+Tdap+bivalent rLP2086) and Group 2 (MCV4+Tdap+saline),before and 1 month after Vaccination 1, for the post-Vaccination 1evaluable immunogenicity population showed robust immune responses amongsubjects after Vaccination 1 for both groups.

Immune Response to MCV4 and Tdap

The GMs of antibodies to Tdap/MCV4 antigens were similar between Group 2(MCV4+Tdap+saline) and Group 1 (MCV4+Tdap+bivalent rLP2086), and theobserved Tdap/MCV4 antigen GMs after Vaccination 1 were indicative of arobust immune response for both groups. RCDCs also supported robustimmune responses after Vaccination 1 for both Group 1 and Group 2.

The noninferiority criteria for MCV4+Tdap+bivalent rLP2086 compared tobivalent rLP2086+saline+saline and MCV4+Tdap+saline required that thelower limit of the 2-sided 95% CIs for the GMRs were greater than 0.67for each of the 10 MCV4/Tdap antigens (1 month after Vaccination 1) andfor each of the hSBA titers using 2 primary MnB test strains (PMB80[A22] and PMB2948 [B24]) (1 month after Vaccination 3). Thisprespecified threshold was met for both MnB test strains and all 10 Tdapand MCV4 antigens. The coprimary immunogenicity objectives of the studywere met as they satisfied the pre-specified non-inferiority criteria.

The immune response induced by MCV4 vaccine and Tdap vaccines givenconcomitantly with bivalent rLP2086 was noninferior to the immuneresponse induced by MCV4 and Tdap vaccines alone when measured 1 monthafter the first vaccination in both groups. The immune response to allantigenic components of the MCV4 and Tdap vaccines were assessed.

The immune response induced by bivalent rLP2086 given concomitantly withMCV4 and Tdap vaccines, as measured by hSBA performed with 2 MnB teststrains was noninferior to the immune response induced by bivalentrLP2086 alone, when measured 1 month after the third vaccination withbivalent rLP2086 in both groups.

For the MCV4+Tdap+bivalent rLP2086 group (Group 1) and theMCV4+Tdap+saline group (Group 2), the estimated differences of theseroresponse rates were between −4.6% and 0.3% for the 6 Tdap and 4 MCV4antigens, with the lower bounds of the 95% CI of the rate differenceequal to 9.1% or less. Since the CIs of the differences were all greaterthan −10%; these differences were not clinically significant. The RCDCsshowed robust immune responses after Vaccination 1 for subjects in bothgroups. Robust GMTs relative to baseline were observed for both groupsthat received MCV4+Tdap.

The 4-fold rise responses to the 2 primary MnB test strains PMB80 (A22)and PMB2958 (B24) from baseline to 1 month after Vaccination 3 weresimilar between the group that received MCV4+Tdap+bivalent rLP2086(84.0% and 85.7%, respectively) and the group that received bivalentrLP2086+saline+saline (88.7% and 87.7%, respectively).

Immunogenicity Discussion

The noninferiority criteria for MCV4+Tdap+bivalent rLP2086 compared tobivalent rLP2086+saline+saline and MCV4+Tdap+saline required that thelower limit of the 2-sided 95% CIs for the GMRs was greater than 0.67(ie, at a 1.5 noninferiority margin) for each of the 10 MCV4/Tdapantigens (1 month after Vaccination 1) and for each of the hSBA titersusing 2 primary MnB test strains (A22 and B24) (1 month afterVaccination 3). This prespecified threshold was met for both MnB teststrains and the 10 Tdap and MCV4 antigens. Thus, both coprimaryimmunogenicity objectives were met. Specifically, these data support atleast the following: 1) The immune response (based on GMs) induced byMCV4 and Tdap vaccines given with bivalent rLP2086 was noninferior tothe immune response induced by MCV4 and Tdap vaccines alone, whenmeasured 1 month after the first vaccination in both groups. The immuneresponse to all components of MCV4 and Tdap vaccines were assessed. 2)The immune response (based on GMT) induced by bivalent rLP2086, asmeasured by hSBA performed with 2 MnB test strains, given with MCV4 andTdap vaccines, was noninferior to the immune response induced bybivalent rLP2086 alone, when measured 1 month after the thirdvaccination with bivalent rLP2086 in both groups.

In this randomized, observer-blinded, active-controlled multicentertrial in subjects aged ≥10 to <13 years, the immunogenicity for bothbivalent rLP2086 and MCV4+Tdap vaccines remained robust when vaccineswere administered concomitantly.

Overall, these data indicate that Tdap, MCV4, MenACWY-CRM, and HPVvaccines may be given concomitantly clinically and these vaccines arerecommended to be given at a single visit. The data from this currentstudy (B1971015) supports that conclusion and also that bivalent rLP2086may be given with Tdap and MCV4. This study showed that at least 2 dosesof bivalent rLP2086 elicited a robust immune response to both primaryMnB test strains. This study also showed that 3 doses of bivalentrLP2086 elicited a robust immune response to both primary MnB teststrains (A22 and B24). The fHBP expressed on these test strains areheterologous to rLP2086 vaccine antigens and represent the mostprevalent subfamily A and subfamily B variants identified among invasivemeningococcal disease isolates in the USA. These 2 variants also areprominent among disease isolates in other geographic regions. Theimmunogenicity assessments for study B1971015 employed definitions of4-fold increases in hSBA titers.

The LOD for each of the 2 primary MnB test strains was a titer equal to1:4 (recognized as the correlate of protection), and the validationLLOQs for these 2 primary MnB test strains were hSBA titers equal to 1:8for PMB2948 (B24) and 1:16 for PMB80 (A22). Prior to receipt of bivalentrLP2086, a low proportion of study subjects had hSBA titers ≥1:4,indicating that most subjects in this population could be consideredunprotected against MnB disease. After the second study vaccination, ahigh proportion of subjects achieved 4-fold increases in hSBA titers andexhibited post-immunization titers ≥1:4 and LLOQ. These response rateswere similar between those subjects receiving coadministration ofbivalent rLP2086 with MCV4+Tdap and those receiving bivalent rLP2086with saline for both MnB strains. The RCDCs also showed that themajority of subjects elicited responses to the 2 primary MnB strains,irrespective of whether bivalent rLP2086 was coadministered withMCV4+Tdap.

In the current study (B1971015), the 4-fold response rates (95% CI)after the second and third dose of bivalent rLP2086 for PMB80 (A22) whenGroup 1 and Group 2 were combined was 64.0% (61.3, 66.5) and 86.3%(84.4, 88.1). Similarly for B24 the 4-fold response rates (95% CI) afterthe second and third dose of bivalent rLP2086 for Group 1 and Group 2combined was 57.4% (54.6, 60.1) and 86.7% (84.8, 88.5).

Of note, the immune responses after the second dose of rLP2086 in studyB1971015 were robust as assessed by both the 4-fold increases in hSBAtiters from baseline and by the proportions of subjects withpost-vaccination hSBA titers ≥LLOQ (1:16 for 22 and 1:8 for B24). ForA22 the percentage (95% CI) of subjects in Groups 1 and 2, combined, whoachieved an hSBA titer ≥LLOQ (1:16) after the second dose was 68.0%(65.4, 70.5). For B24, 64.2% (61.5, 66.8) of subjects achieved an hSBAtiter LLOQ (1:8) after second study dose. These data show substantialhSBA responses to 2 doses of bivalent rLP2086.

Overall, these results indicate that the immune response to MCV4+Tdapand the immune response to bivalent rLP2086 remain robust when bivalentrLP2086 and MCV4+Tdap are administered concomitantly and satisfied thecoprimary study objectives.

These data in subjects aged ≥10 to <13 years satisfied the coprimarystudy objectives, supporting the thesis that bivalent rLP2086 can begiven concomitantly with MCV4+Tdap. Moreover, high proportions ofsubjects achieved hSBA 4-fold responses and hSBA titers ≥LLOQ aftervaccination with 2 or 3 doses of bivalent rLP2086 and these robustresponses were not impacted by concomitant use of MCV4+Tdap. Whenconsidering the immunogenicity and the safety data together, thebenefit-risk profile supports the administration of bivalent rLP2086either alone or with MCV4+Tdap.

Example 12: A Phase 3, Randomized, Active Controlled, Observer BlindedTrial to Assess the Safety and Tolerability of a Meningococcal SerogroupB Bivalent Recombinant Lipoprotein (rLP2086) Vaccine Given in HealthySubjects Aged ≥10 to <26 Years

This was a Phase 3, randomized, active-controlled, observer-blinded,multicenter trial designed to assess the safety and tolerability ofbivalent rLP2086 in healthy subjects aged ≥10 to <26 years.Approximately 5,700 subjects were to be randomly assigned to 1 of 2groups in a 2:1 ratio. Group 1 received bivalent rLP2086 at Month 0(Day 1) followed by subsequent vaccinations at Months 2 and 6. Group 2received a hepatitis A virus (HAV) vaccine at Month 0 and Month 6 and asaline injection at Month 2.

The Bivalent rLP2086 vaccine was in a 0.5 mL dose formulated to contain60 μg each of a purified subfamily A rLP2086 protein (variant A05, SEQID NO: 1) and a purified subfamily B rLP2086 protein (variant B01, SEQID NO: 2), 0.15 M sodium chloride, 2.8 molar ratio polysorbate 80, and0.25 mg of Al³⁺ as aluminum phosphate (AlPO₄) in 10 mMhistidine-buffered saline at pH 6.0.

The HAV vaccine (HAVRIX, 0.5-mL dose or 1.0-mL dose) contained 720 ELISAunits (EL.U.) or 1440 EL.U. of viral antigen. In particular, HAVRIX(Hepatitis A Vaccine) is a sterile suspension of inactivated virus forintramuscular administration. The virus (strain HM175) is propagated inMRC-5 human diploid cells. Age-specific doses of HAV vaccine (HAVRIX)were supplied according to country-specific guidelines. Each 1-mL adultdose of vaccine contains 1440 EL.U. of viral antigen, adsorbed on 0.5 mgof aluminum as aluminum hydroxide. Each 0.5-mL pediatric dose of vaccinecontains 720 EL.U. of viral antigen, adsorbed onto 0.25 mg of aluminumas aluminum hydroxide. HAVRIX contains the following excipients: Aminoacid supplement (0.3% w/v) in a phosphate-buffered saline solution andpolysorbate 20 (0.05 mg/mL). From the manufacturing process, HAVRIX alsocontains residual MRC-5 cellular proteins (not more than 5 mcg/mL),formalin (not more than 0.1 mg/mL), and neomycin sulfate (not more than40 ng/mL), an aminoglycoside antibiotic included in the cell growthmedia. HAVRIX is formulated without preservatives.

Sterile normal saline solution (0.9% sodium chloride) for injection, wassupplied as a 0.5-mL dose.

TABLE A Study Design Final Telephone Telephone Telephone TelephoneFollow-up Telephone Vax 1 Contact Vax 2 Contact Contact Contact Vax 3Visit Contact Visit 1 2 3 4 5 6 7 8  9 Number Approx 0 1 2 3 4 5 6 7 12month Group 1 rLP2086 rLP2086 rLP2086 (N = 3800) Group 2 HAV Normal HAV(N = 1900) vaccine saline vaccine Abbreviations: approx = approximate;Vax = vaccinnation.This study assessed the safety and tolerability of bivalent rLP2086 atthe 120-μg dose level administered in healthy subjects aged ≥10 to <26years using a 0-, 2-, and 6-month schedule and provided an importantcomponent of the safety database for bivalent rLP2086.

HAV vaccine administered at Months 0 and 6 was chosen as the control inthis study because there was no proven safe, immunogenic, and effectivevaccine against MnB that could serve as an active control. In comparisonto other recommended vaccines for this age group, HAV vaccine has awell-established tolerability profile. In addition, HAV vaccineconferred a benefit to subjects who may become at increased risk forhepatitis A viral infection either during future travel or otherexposures. Saline was administered at Month 2 in the control group tomaintain the study blind. Subjects were ineligible to participate ifthey met any of the exclusion criteria, including: Previous vaccinationwith any meningococcal serogroup B vaccine; Subjects who had receivedprior HAV vaccination; Contraindication to vaccination with any HAVvaccine. Subjects received 1 dose of 120 μg of bivalent rLP2086 or HAVvaccine/saline/HAV vaccine at each of the vaccination visits (Visits 1,3, and 7) according to Table A.

Subjects received vaccinations using a 0-, 2-, 6-month schedule. Group 1received bivalent rLP2086 at Month 0 (Day 1) followed by subsequentvaccinations at Months 2 and 6. Group 2 received HAV vaccine at Month 0and Month 6 and an injection with saline at Month 2 to maintain thestudy blind. Age-specific doses of HAV vaccine were supplied accordingto country-specific guidelines.

The primary endpoints for the safety population were the percentage ofsubjects with at least 1 serious adverse event (SAE) that occurredduring the time period from the first study vaccination through 6 monthsafter the last study vaccination and the percentage of subjects with atleast 1 medically attended adverse event (AE) that occurred during thetime period within 30 days after each vaccination.

Overall, the proportion of subjects reporting at least 1 SAE within 30days of each vaccination was ≤3.0% for both groups and similar betweenbivalent rLP2086 and HAV vaccine/saline control. Throughout the study, anumerically greater proportion of SAEs were reported by subjects inGroup 2 (HAV vaccine/saline) than Group 1 (bivalent rLP2086) (2.52% and1.55%, respectively). SAEs were also reported by a numerically largerproportion of subjects in Group 2 compared to Group 1 during thevaccination and follow-up phases. Overall, the percentage of AEsreported during the vaccination phase was higher in Group 1 compared toGroup 2. Subjects in Group 1 reported more reactogenicity AEs thansubjects in Group 2 during the vaccination phase. Reactogenicity AEswere defined as any AE with an onset within 7 days after vaccination andmatched a predefined preferred term list. No new safety concerns wereidentified in this study for the group that received bivalent rLP2086.The data from this study demonstrate that bivalent rLP2086 is safe andwell tolerated in subjects aged ≤0 to <26 years.

Example 13: Immunogenicity of Meningococcal Serogroup B Bivalent rLP2086Vaccine in Healthy Adolescents Given Concomitantly with Other LicensedVaccines Background:

Bivalent rLP2086 (TRUMENBA®) a N. meningitidis serogroup B (MnB) factorH binding protein (fHBP) vaccine, is approved in the US for preventionof invasive MnB disease in 10-25 years olds. Concomitant administrationof recommended vaccines in this population improves compliance andmaximizes public health impact.

Objective:

To demonstrate noninferiority of immune responses (based on geometricmean [GM] titers) induced by GARDASIL® (HPV4), or MENACTRA®(meningococcal Groups A, C, Y and W-135, MCV4) and ADACEL® (Tdap), whengiven concomitantly with TRUMENBA.

Design/Methods:

Study 1: subjects (11-<18 years) received TRUMENBA+HPV4, TRUMENBA, orHPV4 at months 0, 2 and 6. Subjects receiving only TRUMENBA or HPV4received saline injections to maintain blinding. Study 2: subjects(10-<13 years) received MCV4+Tdap+TRUMENBA, MCV4+Tdap, or TRUMENBA.TRUMENBA was administered at months 0, 2 and 6. MCV4 and Tdap were givenat month 0. Groups receiving only MCV4 and Tdap, or TRUMENBA receivedsaline injections at months 0, 2, and 6 to maintain blinding. Immuneresponses were assessed after final vaccine administration. In bothstudies, noninferiority was achieved if the lower bound of the 95% CI ofthe GM ratio between groups was >0.67 (a 1.5-fold noninferioritymargin).

Results:

The noninferiority criterion was met for all HPV antigens except HPV18(lower 95% CI=0.62) and for all Tdap and MCV4 antigens when thesevaccines were coadministered with TRUMENBA. Seroconversion rates for allHPV antigens were ≥99% when HPV4 was given alone or with TRUMENBA. Thenoninferiority criterion was met for MnB hSBA responses when TRUMENBAwas coadministered with licensed vaccines (Table).

Data support coadministration of TRUMENBA with HPV4, MCV4, and Tdapvaccines recommended for US adolescents.

TABLE Comparison of Geometric Means at 1 Month After Last VaccinationN^(a) GM^(b) N^(a) GM^(b) N^(a) GM^(b) Ratio^(c) (95% CI)^(d) Study 1Bivalent Bivalent rLP2086 + rLP2086 + Saline + HPV Antigens^(e) HPV4Saline HPV4 (Group 1 vs Group 3) (Group 1) (Group 2) (Group 3) HPV-6 813451.8 NA NA 423 550.3 0.82 (0.72, 0.94) HPV-11 813 892.9 NA NA 4231084.3 0.82 (0.74, 0.91) HPV-16 813 3695.4 NA NA 423 4763.4 0.78 (0.68,0.88) HPV-18 813 744.0 NA NA 423 1047.4 0.71 (0.62, 0.81) hSBA MnBStrains [Variants]^(f) (Group 1 vs Group 2) PMB80 [A22] 803 53.3 80157.8 NA NA 0.92 (0.85, 1.00) PMB2948 [B24] 788 25.8 793 28.0 NA NA 0.92(0.84, 1.01) Study 2 Saline + MCV4 + Tdap + Saline + Bivalent MCV4 +Tdap + Bivalent Tdap Antigens^(g) rLP2086 Saline rLP2086 (Group 1 vsGroup 2) (Group 1) (Group 2) (Group 3) Diphtheria toxoid 778 9.3 780 9.8NA NA 0.94 (0.86, 1.03) Tetanus toxoid 778 9.4 780 10.3 NA NA 0.92(0.85, 0.99) Pertussis toxoid 778 13.2 780 14.2 NA NA 0.93 (0.85, 1.02)Pertussis filamentous 778 112.0 780 122.9 NA NA 0.91 (0.84, 0.98)hemagglutinin Pertussis pertactin 778 202.0 780 228.9 NA NA 0.88 (0.80,0.98) Pertussis fimbriae 778 138.1 780 154.2 NA NA 0.90 (0.74, 1.08)agglutinogens types 2 + 3 rSBA MCV4 Antigens^(h) (Group 1 vs Group 2)Serogroup A 763 4647.3 772 5113.0 NA NA 0.91 (0.82, 1.01) Serogroup C768 1679.2 767 1650.2 NA NA 1.02 (0.90, 1.15) Serogroup Y 771 2212.6 7702244.9 NA NA 0.99 (0.89, 1.09) Serogroup W-135 751 5925.1 765 6367.9 NANA 0.93 (0.83, 1.04) hSBA MnB Strains^(f) [Variant] (Group 1 vs Group 3)PMB80 [A22] 679 45.9 NA NA 674 49.7 0.92 (0.84, 1.02) PMB2948 [B24] 67024.8 NA NA 656 27.4 0.90 (0.82, 1.00) CI = confidence interval; GMT =geometric mean titer; HPV = human papillomavirus, HPV4 = humanpapillomavirus quadrivalent vaccine; MCV4 = meningococcal (Groups A, C,Y and W-135) polysaccharide diphtheria toxoid conjugate vaccine; MnB =Neisseria meningitidis serogroup B; NA = not applicable; Tdap = tetanustoxoid, reduced diphtheria toxoid and acellular pertussis vaccineadsorbed. ^(a)N = number of subjects with valid and determinate assayresults for the given antigen or strain. ^(b)Geometric means werecalculated using all subjects with valid and determinate assay resultsat 1 month after Vaccination 3. ^(c)Ratios of GMs. ^(d)ConfidenceIntervals (CIs) for the ratio are back transformations of a confidenceinterval based on the Student t distribution for the mean difference ofthe logarithms of the measures (Group 1 - Group 3 for HPV titers; Group1 - Group 2 for Tdap/MCV4 antigens). ^(e)Immune responses to HPVantigens assessed in a competitive LUMINEX immunoassay 1 month after thethird Gardasil ® vaccination. ^(f)Immune responses to MnB antigensassessed in a serum bactericidal assay using human complement 1 monthafter the third bivalent rLP2086 vaccination. ^(g)Immune responses todiphtheria, tetanus, and pertussis antigens assessed in a LUMINEX assay1 month after the single ADACEL ® vaccination. ^(h)Immune responses toMCV4 antigens assessed in a serum bactericidal assay using rabbitcomplement 1 month after the single MENACTRA ® vaccination.

Example 14: Phase 3 Trial of Immunogenicity of Bivalent rLP2086, aMeningococcal Serogroup B Vaccine, in Adolescents: Bactericidal ActivityAgainst a Panel of Antigenically Diverse Strains Background:

Bivalent rLP2086, which targets factor H binding proteins (fHBP), isapproved in the US to prevent meningococcal serogroup B (MnB) disease in10-25-year-olds. Broad protection with bivalent rLP2086 was initiallydemonstrated in hSBAs with 4 diverse invasive MnB strains expressingfHBPs with sequences different from vaccine antigens. In this pivotalphase 3 trial, broad coverage against MnB disease is further supportedby hSBA data with 10 additional MnB test strains representing thediversity of circulating invasive MnB strains (ClinicalTrials.gov:NCT01830855).

Methods:

Healthy subjects aged 10-<19 years were randomized to receive bivalentrLP2086 at 0, 2, and 6 months, or hepatitis A virus vaccine at 0 and 6months and saline at 2 months. Immune responses were assessed in hSBAswith 4 primary MnB test strains (primary endpoint; N=1210-1266) and in apopulation subset using 10 secondary MnB test strains (secondaryendpoint; N=266-281). All strains expressed vaccine-heterologous fHBP.

Results:

hSBA responses 1 month after doses 2 and 3 among bivalent rLP2086recipients against 4 primary MnB test strains measured by hSBA titers≥LLOQ were 64.0%-99.1% and 87.1%-99.5%, respectively (Table). hSBAresponses to 10 secondary MnB test strains were 61.1%-100.0% and75.1%-98.6% 1 month after dose 2 and 3, respectively. hSBA GMTs for eachsecondary test strain increased from 4.5-11.4 at baseline to 22.1-93.5after dose 3.

TABLE Immunogenicity of bivalent rLP2086 against secondary MnB teststrains Strain hSBA titers ≥ LLOQ, % (1:8 or 1:16) hSBA GMTs (fHBPVariant) N Baseline After dose 2* After dose 3 Baseline After dose 3Primary strains PMB80 (A22) 1266 33.2 94.3 97.8 12.6 86.8 PMB2001 (A56)1229 27.5 99.1 99.5 8.4 222.5 PMB2948 (B24) 1250 6.4 66.4 87.1 4.5 24.1PMB2707 (B44) 1210 3.6 64.0 89.3 4.3 50.9 Secondary strains PMB3175(A29) 278 17.5 100.0 98.6 5.7 93.5 PMB3010 (A06) 280 9.4 84.0 95.7 9.378.6 PMB3040 (A07) 280 43.1 93.8 96.4 11.4 63.5 PMB824 (A12) 277 3.967.4 75.1 8.4 22.3 PMB1672 (A15) 266 20.7 65.6 87.2 5.9 31.0 PMB1989(A19) 275 11.3 84.5 92.7 9.1 57.6 PMB1256 (B03) 279 4.3 61.1 92.5 4.551.7 PMB866 (B09) 276 15.2 76.3 86.2 5.2 22.9 PMB431 (B15) 281 28.7 96.898.2 7.3 47.7 PMB648 (B16) 278 7.6 61.6 81.7 4.7 22.1 *For the secondarystrains, subset of subjects; n = 86-97. LLOQ = 1:16 for A06, A12, A19,and A22; 1:8 for A07, A15, A29, A56, B03, B09, B15, B16, B24, and B44.

Conclusions:

Bivalent rLP2086 vaccination resulted in robust immune responses againstdiverse MnB strains heterologous to vaccine antigens after 2 and 3doses. A high proportion of individuals developed protective hSBA titresgreater than the correlate of protection (hSBA titre ≥1:4) to 10additional MnB test strains. Collectively, these phase 3 immunogenicitydata support the broad protection afforded by bivalent rLP2086 againstMnB disease in adolescents.

Example 15: Phase 3 Trial of Immunogenicity of Bivalent rLP2086, a

Meningococcal Serogroup B Vaccine, in Young Adults: BactericidalActivity Against a Panel of Antigenically Diverse Strains

BACKGROUND

Bivalent rLP2086, which targets factor H binding proteins (fHBP), isapproved in the US to prevent meningococcal serogroup B (MnB) disease in10-25-year-olds. Broad protection with bivalent rLP2086 was initiallydemonstrated in hSBAs with 4 diverse invasive MnB strains expressingfHBPs with sequences different from vaccine antigens. In this pivotalphase 3 trial, broad coverage against MnB disease is further supportedby hSBA data with 10 additional MnB test strains representing thediversity of circulating invasive MnB strains (ClinicalTrials.gov:NCT01352845).

Methods:

Healthy subjects aged 18-<26 years were randomized to receive bivalentrLP2086 or saline at 0, 2, and 6 months. Immune responses were assessedin hSBAs with 4 primary MnB test strains (primary endpoint; N=1702-1714)and in a population subset (N=273-284) using 10 secondary MnB teststrains (secondary endpoint; N=273-284). All strains expressedvaccine-heterologous fHBP.

Results:

hSBA responses 1 month after dose 2 and 3 among bivalent rLP2086recipients against 4 primary MnB test strains as measured by hSBA titers≥LLOQ were 68.3%-97.4% and 87.4%-99.4%, respectively (Table). hSBAresponses to 10 secondary MnB test strains were 51.6%-97.9% and71.3%-99.3% 1 month after dose 2 and 3, respectively. hSBA GMTs for eachsecondary strain increased from 5.1-13.9 at baseline to 20.6-96.3 afterdose 3.

TABLE Immunogenicity of bivalent rLP2086 against secondary MnB teststrains Strain hSBA titers ≥ LLOQ, % (1:8 or 1:16) hSBA GMTs (fHBPVariant) N Baseline After dose 2* After dose 3 Baseline After dose 3Primary strains PMB80 (A22) 1714 33.6 84.7 93.5 12.8 74.3 PMB2001 (A56)1709 32.2 97.4 99.4 8.8 176.7 PMB2948 (B24) 1702 33.1 86.5 95.1 7.6 49.5PMB2707 (B44) 1703 11.0 68.3 87.4 4.8 47.6 Secondary strains PMB3175(A29) 283 31.1 96.8 99.3 7.1 96.3 PMB3010 (A06) 275 16.0 77.8 92.0 10.369.9 PMB3040 (A07) 277 55.8 97.9 95.7 13.9 60.4 PMB824 (A12) 275 5.057.6 71.3 8.4 20.6 PMB1672 (A15) 279 37.3 83.2 91.8 8.0 43.1 PMB1989(A19) 284 28.8 87.4 95.8 12.1 87.3 PMB1256 (B03) 273 11.2 57.9 86.4 5.149.8 PMB866 (B09) 274 23.5 65.3 77.0 6.1 23.3 PMB431 (B15) 276 43.8 86.596.7 9.1 49.4 PMB648 (B16) 273 21.9 51.6 78.0 6.2 26.5 *For thesecondary strains, subset of subjects; n = 90-96. LLOQ = 1:16 for A06,A12, A19, and A22; 1:8 for A07, A15, A29, A56, B03, B09, B15, B16, B24,and B44.

Conclusions:

Bivalent rLP2086 vaccination elicited robust immune responses againstdiverse MnB strains expressing fHBP variants heterologous to vaccineantigens after 2 and 3 doses. A high proportion of individuals developedprotective hSBA titres greater than the correlate of protection (hSBAtitre ≥1:4) to 10 additional MnB test strains. Collectively, these phase3 immunogenicity data support the broad protection afforded by bivalentrLP2086 against MnB disease in young adults.

What is claimed is:
 1. A method for inducing a bactericidal immuneresponse against a Neisseria meningitidis serogroup B subfamily Astrain, and against a Neisseria meningitidis serogroup B subfamily Bstrain in a human subject, comprising administering an effective amountof a N. meningitidis rLP2086 composition wherein said N. meningitidisrLP2086 composition comprises a) a first lipidated polypeptidecomprising the amino acid sequence set forth in SEQ ID NO: 1, and b) asecond lipidated polypeptide comprising the amino acid sequence setforth in SEQ ID NO: 2; wherein the immune response is i) bactericidalagainst a N. meningitidis serogroup B subfamily A strain that isheterologous to a N. meningitidis strain expressing A05, and is ii)bactericidal against a N. meningitidis serogroup B subfamily B strainthat is heterologous to a N. meningitidis strain expressing B01.
 2. Themethod according to claim 1, wherein the immune response is bactericidalagainst a N. meningitidis serogroup B subfamily A strain that isheterologous to N. meningitidis strain M98250771.
 3. The methodaccording to claim 1, wherein the immune response is bactericidalagainst a N. meningitidis serogroup B subfamily B strain that isheterologous to N. meningitidis strain CDC1127.
 4. The methodaccordingly to claim 1, wherein the immune response is bactericidalagainst a N. meningitidis serogroup B subfamily B strain that isheterologous to N. meningitidis strain CDC1573.
 5. The method accordingto claim 1, wherein the immune response is bactericidal against at leastone N. meningitidis serogroup B strain that expresses fHBP selectedfrom: A22 fHBP; A56 fHBP; B24 fHBP; and B44 fHBP.
 6. The methodaccording to claim 1, wherein the immune response is bactericidalagainst at least one N. meningitidis serogroup B strain that expressesfHBP selected from: A29 fHBP; A06 fHBP; A07 fHBP; A12 fHBP; A15 fHBP;A19 fHBP; B03 fHBP; B09 fHBP, B15 fHBP, and B16 fHBP.
 7. The methodaccording to claim 1, wherein the N. meningitidis rLP2086 compositionfurther comprises polysorbate-80.
 8. The method according to claim 7,wherein the N. meningitidis rLP2086 composition further comprisesaluminum.
 9. The method according to claim 8, wherein the N.meningitidis rLP2086 composition further comprises histidine buffer. 10.The method according to claim 9, wherein the N. meningitidis rLP2086composition further comprises sodium chloride.
 11. The method accordingto claim 1, wherein the N. meningitidis rLP2086 composition comprisesabout 120 μg/mL of the first polypeptide; about 120 μg/mL of the secondpolypeptide; about 2.8 molar ratio of polysorbate-80; about 0.5 mg/mLaluminum; about 10 mM histidine; and about 150 mM sodium chloride. 12.The method according to claim 1, wherein the N. meningitidis rLP2086composition comprises about 60 μg of the first polypeptide; about 60 μgof the second polypeptide; about 18 μg polysorbate-80; about 250 μgaluminum; about 780 μg histidine; and about 4380 μg sodium chloride. 13.The method according to claim 1, wherein the effective amount of the N.meningitidis rLP2086 composition comprises one dose.
 14. The methodaccording to claim 1, wherein the effective amount of the N.meningitidis rLP2086 composition comprises two doses.
 15. The methodaccording to claim 14, wherein the effective amount of the N.meningitidis rLP2086 composition further comprises a booster dose. 16.The method according to claim 1, wherein the effective amount of the N.meningitidis rLP2086 composition comprises at most three doses.